Macrocyclic compounds and uses thereof

ABSTRACT

The present invention provides novel compounds (e.g., compounds of Formulae (I), (II), (III), (IV)) having tumor vascular remodeling effect and/or anti-CAF (Cancer Associated Fibroblasts) activity, or pharmaceutically acceptable salts thereof, optionally in a pharmaceutically acceptable carrier, and a medical uses thereof.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. § 371 ofInternational PCT Application, PCT/US2018/061250, filed Nov. 15, 2018,which claims priority under 35 U.S.C. § 119(e) to U.S. ProvisionalPatent applications, U.S. Ser. No. 62/586,416, filed Nov. 15, 2017; andU.S. Ser. No. 62/765,310, filed Aug. 20, 2018; the entire contents ofeach of which is incorporated herein by reference.

TECHNICAL FIELD

The present invention provides novel macrocyclic compounds having tumorvascular remodeling effects and anti-CAF (Cancer Associated Fibroblast)activity. The compounds can be used for, e.g., treating cancer orinhibiting tumor growth in a subject.

BACKGROUND

Halichondrins, such as Halichondrin B, are anticancer agents originallyisolated from the marine sponge Halichondria okadai (See, e.g., D.Uemura et al. “Norhalichondrin A: An Antitumor Polyether Macrolide froma Marine Sponge” J. Am. Chem. Soc., 107, 4796 (1985)), and subsequentlyfound in Axinella sp., Phakellia carteri, and Lissodendoryx sp. A totalsynthesis of Halichondrin B was published in 1992 (See, e.g., Y Kishi etal. “Total Synthesis of Halichondrin B and Norhalichondrin B” J. Am.Chem. Soc., 114, 3162 (1992)).

Halichondrin B has demonstrated in vitro inhibition of tubulinpolymerization, microtubule assembly, beta 5-tubulin crosslinking, GTPand vinblastine binding to tubulin, and tubulin-dependent GTPhydrolysis, and has shown in vitro and in vivo anti-cancer properties(See, e.g., Y Hirata et al. “Halichondrins-antitumor polyethermacrolides from a marine sponge” Pure Appl. Chem., 58, 701 (1986);Fodstad et al. “Comparative antitumor activities of halichondrins andvinblastine against human tumor xenografts” J. of ExperimentalTherapeutics & Oncology 1996; 1: 119, 125).

Eribulin mesylate (Halaven™), which was developed based on HalichondrinB (See, e.g., International Publication No. WO 1999/065894, publishedDec. 23, 1999; International Publication No. WO 2005/118565, publishedDec. 15, 2005; and W. Zheng et al. “Macrocyclic ketone analogues ofhalichondrin B” Bioorganic & Medicinal Chemistry Letters 14, 5551-5554(2004)), is currently in clinical use in many countries for thetreatment of, e.g., metastatic breast cancer and advanced liposarcoma.

Additional patent publications describing Halichondrins include U.S.Pat. No. 5,436,238 to Kishi, et al., issued Jul. 25, 1995; U.S. Pat. No.5,338,865 to Kishi, et al., issued Aug. 16, 1994; and WO 2016/003975filed by Kishi, et al., all of which are assigned to the President andFellows of Harvard College.

See also, e.g., U.S. Pat. Nos. 5,786,492; 8,598,373; 9,206,194;9,469,651; WO/2009/124237A1; WO/1993/017690A1; WO/2012/147900A1; U.S.Pat. Nos. 7,982,060; 8,618,313; 9,303,050; 8,093,410; 8,350,067;8,975,422; 8,987,479; 8,203,010; 8,445,701; 8,884,031; RE45,324;8,927,597; 9,382,262; 9,303,039; WO/2009/046308A1; WO/2006/076100A3;WO/2006/076100A2; WO/2015/085193A1; WO/2016/176560A1; U.S. Pat. Nos.9,278,979; 9,029,573; WO/2011/094339A1; WO/2016/179607A1;WO/2009/064029A1; WO/2013/142999A1; WO/2015/066729A1; WO/2016/038624A1;and WO/2015/000070A1.

Cancer associated fibroblasts (CAFs), which are widely found in avariety of solid tumors, are stromal cells. It is well known that CAFsplay an important role in angiogenesis, invasion, and metastasis. Thereis a close correlation between the amount of CAFs and clinical prognosisin, for example, invasive breast cancer (See, e.g., M. Yamashita et al.“Role of stromal myofibroblasts in invasive breast cancer: stromalexpression of alpha-smooth muscle actin correlates with worse clinicaloutcome” Breast Cancer 19, 170, 2012) and esophageal adenocarcinoma(See, e.g., T. J. Underwood et al. “Cancer-associated fibroblastspredict poor outcome and promote periostin-dependent invasion inesophageal adenocarcinoma” Journal of Pathol., 235, 466, 2015). It hasalso been reported that CAFs correlate to resistance in a variety oftumors, such as, for example, breast cancer (See, e.g., P. Farmer et al.“A stroma-related gene signature predicts resistance to neoadjuvantchemotherapy in breast cancer” Nature Medicine., 15(1), 68, 2009), andhead and neck cancer (See, e.g., S. Schmitz et al. “Cetuximab promotesepithelial to mesenchymal transition and cancer associated fibroblastsin patients with head and neck cancer” Oncotarget, 6 (33), 34288, 2015;Y Matsuoka et al. “The tumor stromal features are associated withresistance to 5-FU-based chemoradiotherapy and a poor prognosis inpatients with oral squamous cell carcinoma” APMIS 123(3), 205, 2015).

It has thus been observed that tumor vascular remodeling effects andanti-CAF activity result in the improvement of the cancermicroenvironment, which assists tumor treatment. Blood vessels areessential for the growth of tumors. Reconstructed blood vessels intumors can deliver anti-cancer agents to the tumors, in addition toalleviating hypoxia. It is reported that eribulin-induced remodeling ofabnormal tumor vasculature leads to a more functional microenvironmentthat may reduce the aggressiveness of tumors due to the elimination ofinner tumor hypoxia. Because abnormal tumor microenvironments enhanceboth drug resistance and metastasis, the apparent ability of eribulin toreverse these aggressive characteristics may contribute to its clinicalbenefits (See, e.g., Y Funahashi et al. “Eribulin mesylate reduces tumormicroenvironment abnormality by vascular remodeling in preclinical humanbreast cancer models” Cancer Sci. 105 (2014), 1334-1342). Anti-cancerdrugs having tumor vascular remodeling effects and anti-CAF activitieshave not been reported as of today.

Despite the progress made, additional compounds are needed to progressresearch and medical care of patients with tumors and cancer.

SUMMARY OF THE INVENTION

Halichondrins, as well as analogs and derivatives thereof, are usefultherapeutic agents. Examples of halichondrins, analogs, and derivativesthereof, as well as methods of using the same, and methods ofsynthesizing the same, can be found in, e.g., U.S. Publication No.2017/0137437, published May 18, 2017; International Publication No. WO2016/003975, published Jan. 7, 2016; U.S. Publication No. 2018/0230164,published Aug. 16, 2018; International Publication No. WO 2016/176560,published Nov. 3, 2016; U.S.

Publication No. 2018/0155361, published Jun. 7, 2018; and U.S. Pat. No.9,938,288, issued Apr. 10, 2018; the entire contents of each of which isincorporated herein by reference.

The present invention relates to macrocyclic compounds (e.g., compoundsof Formulae (I), (II), (III), and (IV)), and pharmaceutically acceptablesalts thereof, and isotopically labeled derivatives thereof, andpharmaceutical compositions thereof. In certain embodiments, thecompounds have tumor vascular remodeling effects and anti-CAF activity.

The invention also provides methods of using the compounds providedherein, e.g., for treating a proliferative disease in a subject. Incertain embodiments, the invention includes methods of using compoundsprovided herein for treating a subject with cancer, methods forreversibly or irreversibly inhibiting mitosis in a cell, and methods forinhibiting tumor growth in vitro, in vivo, or in a subject. In anotheraspect, the present invention provides kits comprising a compoundprovided herein, or a pharmaceutically acceptable salt thereof, or apharmaceutical composition thereof.

In one aspect, the present invention provides compounds of Formula (I):

and pharmaceutically acceptable salts and isotopically labeledderivatives thereof, wherein:

R^(N1) and R^(N2) are independently hydrogen, optionally substitutedalkyl, optionally substituted acyl, or a nitrogen protecting group,optionally wherein R^(N1) and R^(N2) are joined together with theintervening atoms to form optionally substituted heterocyclyl oroptionally substituted heteroaryl;

R^(P1) is hydrogen, optionally substituted alkyl, optionally substitutedacyl, or an oxygen protecting group;

R^(X) is hydrogen or —OR^(Xa);

R^(Y) is hydrogen or —OR^(Ya);

R^(Xa) and R^(Ya) are independently hydrogen, optionally substitutedalkyl, optionally substituted acyl, or an oxygen protecting group,optionally wherein R^(Xa) and R^(Ya) are joined together with theintervening atoms to form optionally substituted heterocyclyl; and

n is 1 or 2.

In certain embodiments, the compound of formula (I) is not thefollowing:

or a pharmaceutically acceptable salt or isotopically labeled derivativethereof.

In another aspect, the present invention provides compounds of Formula(II):

and pharmaceutically acceptable salts and isotopically labeledderivatives thereof, wherein:

R^(N1) and R^(N2) are independently hydrogen, optionally substitutedalkyl, optionally substituted acyl, or a nitrogen protecting group,optionally wherein R^(N1) and R^(N2) are joined together with theintervening atoms to form optionally substituted heterocyclyl oroptionally substituted heteroaryl;

R^(P1) and R^(P2) are independently hydrogen, optionally substitutedalkyl, optionally substituted acyl, or an oxygen protecting group;

R^(X) is hydrogen or —OR^(Xa);

R^(Y) is hydrogen or —OR^(Ya); R^(Xa) and R^(Ya) are independentlyhydrogen, optionally substituted alkyl, optionally substituted acyl, oran oxygen protecting group, optionally wherein R^(Xa) and R^(Ya) arejoined together with the intervening atoms to form optionallysubstituted heterocyclyl; and

m is 0 or 1.

In another aspect, the present invention provides compounds of Formula(III):

and pharmaceutically acceptable salts and isotopically labeledderivatives thereof, wherein:

R^(O) is hydrogen, optionally substituted alkyl, optionally substitutedacyl, or an oxygen protecting group;

R^(P1) is hydrogen, optionally substituted alkyl, optionally substitutedacyl, or an oxygen protecting group;

R^(X) is hydrogen or —OR^(Xa);

R^(Y) is hydrogen or —OR^(Ya);

R^(Xa) and R^(Ya) are independently hydrogen, optionally substitutedalkyl, optionally substituted acyl, or an oxygen protecting group,optionally wherein R^(Xa) and R^(Ya) are joined together with theintervening atoms to form optionally substituted heterocyclyl; and

n is 1 or 2.

In certain embodiments, the compound of Formula (III) is not thefollowing:

or a pharmaceutically acceptable salt or isotopically labeled derivativethereof.

In yet another aspect, the present invention provides compounds ofFormula (IV):

and pharmaceutically acceptable salts and isotopically labeledderivatives thereof, wherein:

R^(O) is hydrogen, optionally substituted alkyl, optionally substitutedacyl, or an oxygen protecting group;

R^(P1) is hydrogen, optionally substituted alkyl, optionally substitutedacyl, or an oxygen protecting group;

R^(X) is hydrogen or —OR^(Xa);

R^(Y) is hydrogen or —OR^(Ya);

R^(Xa) and R^(Ya) are independently hydrogen, optionally substitutedalkyl, optionally substituted acyl, or an oxygen protecting group,optionally wherein R^(Xa) and R^(Ya) are joined together with theintervening atoms to form optionally substituted heterocyclyl.

In one aspect, the invention features a compound which is Compound (1):

and pharmaceutically acceptable salts thereof, and isotopically labeledderivatives thereof. In certain embodiments, Compound (1) is excludedfrom the invention. In certain embodiments, Compound (1) and allpharmaceutically acceptable salts thereof and isotopically labeledderivatives thereof are excluded from the invention.

In another aspect, the invention provides pharmaceutical compositionscomprising a compound provided herein, or a pharmaceutically acceptablesalt or isotopically labeled derivative thereof. The pharmaceuticalcompositions may comprise one or more pharmaceutically acceptableexcipients or carriers. The pharmaceutical compositions may furthercomprise one or more additional therapeutic agents in combination,alternation, or other kind of synchronized therapy, to achieve thedesired goal of treatment.

The invention also features methods of making compounds provided herein,or intermediates thereto. The synthetic intermediates are also providedherein as part of the invention.

It has been discovered that compounds provided herein have anadvantageous effect on tumor vascular remodeling and has anti-CAFactivity, as demonstrated in the Figures and Examples. Accordingly, thecompounds provided herein have potential use in the treatment ofproliferative diseases. In certain embodiments, the compounds can beused to treat cancer (e.g., squamous cell carcinoma of the head and neck(SCCHN), breast cancer, esophageal cancer, uterine cancer, ovariancancer, colorectal cancer, endometrial cancer, gastric cancer, lungcancer, small bowel cancer, bladder cancer, sweat gland cancer,sarcomas, rare cancers).

In another aspect, the present invention provides methods for treatingcancer or inhibiting tumor growth in a subject with a compound providedherein, or a pharmaceutically acceptable salt, or isotopically labeledderivative thereof. In certain embodiments, the present inventionprovides methods for inhibiting any tumor growth or cancer that willrespond to a compound with tumor vascular remodeling effects and/oranti-CAF activity, in a subject, typically a human, with a compoundprovided herein, or a pharmaceutically acceptable salt, or isotopicallylabeled derivative thereof.

A compound provided herein, or a pharmaceutically acceptable salt, orisotopically labeled derivative thereof, or a composition thereof, maybe administered in combination with any other active agent that providesbeneficial results for the patient. In one embodiment, a compoundprovided herein is used in combination, alternation, or othersynchronized therapy with an immunotherapy. In certain embodiments, theimmunotherapy is an anti-EGFR (epidermal growth factor receptor)antibody, an anti-HER2 (human epidermal growth factor receptor)antibody, an anti-PD-1 antibody, or an anti-PD-L1 antibody, as describedin more detail below.

For example, a method is provided to treat squamous cell carcinoma ofthe head and neck (SCCHN) in a subject, typically a human, in needthereof comprising administering to the subject an effective amount of acompound provided herein, or a pharmaceutically acceptable salt, orisotopically labeled derivative thereof, or a composition thereof, incombination with an anti-EGFR (epidermal growth factor receptor) mAbtherapy. In certain embodiments, the anti-EGFR (epidermal growth factorreceptor) mAb is cetuximab.

As another example, a method to treat breast cancer in a subject,typically a human, in need thereof comprising administering to saidsubject an effective amount of a compound provided herein, or apharmaceutically acceptable salt, or isotopically labeled derivativethereof, or a composition thereof, in combination with an HER2 (humanepidermal growth factor receptor) mAb therapy. In certain embodiments,the HER2 (human epidermal growth factor receptor) mAb is trastuzumab. Inother embodiments, the compound may be used to treat breast cancer incombination with traditional chemotherapy, such as adriamycin,cyclophosphamide, taxol, etc., or an anti-estrogen such as a selectiveestrogen modulator (SERM), a selective estrogen degrader (SERD), apartial or total estrogen inhibitor (such as fulvestrant) or a CDK 4/6inhibitor such as palbociclib (Pfizer).

Another aspect of the present invention provides a compound of Formula(I), (II), (III), or (IV), or a pharmaceutically acceptable salt, orisotopically labeled derivative thereof, which may be in the form of ahydrate, solvate, polymorph, stereoisomer, or a composition thereof, ina kit, which may be a dosage form package. The kits described herein mayinclude a single dose or multiple doses of the compound orpharmaceutical composition thereof. A kit of the invention may includeinstructions for using the provided therapeutic dosage forms (e.g.,instructions for using the compound or pharmaceutical compositionincluded in the kit).

The present invention thus includes at least the following features:

-   -   (i) A compound of Formula (I), (II), (III), or (IV), or a        pharmaceutically acceptable salt or isotopically labeled        derivative thereof, which may be optionally be in the form of a        hydrate, solvate, stereoisomer, or polymorph;    -   (ii) A method for treatment that includes administering an        effective amount to a subject such as a human of a compound        provided herein, or a pharmaceutically acceptable salt or        isotopically labeled derivative, which may be optionally be in        the form of a hydrate, solvate, stereoisomer, or polymorph, to        treat a proliferative disease. In certain embodiments, the        proliferative disease is cancer. In certain embodiment, the        cancer is head and neck cancer (e.g., squamous cell carcinoma of        the head and neck (SCCHN), adenoid cystic carcinoma), breast        cancer, esophageal cancer (e.g., esophageal adenocarcinoma),        uterine cancer (e.g., uterine sarcoma), ovarian cancer,        colorectal cancer, or sarcoma (e.g., synovial sarcoma,        angiosarcoma, soft tissue sarcoma, fibrosarcoma, uterine        sarcoma, intimal sarcoma). In certain embodiments, the cancer is        bladder cancer (e.g., urothelial carcinoma), gastric cancer,        small bowel cancer (e.g., small bowel adenocarcinoma),        endometrial cancer, lung cancer (e.g., non-small cell lung        cancer), or sweat gland cancer (e.g., sweat gland carcinoma). In        certain embodiments, the cancer is a rare cancer;    -   (iii) A method for treatment that includes administering an        effective amount to a subject such as a human of a compound        provided herein, or a pharmaceutically acceptable salt or        isotopically labeled derivative thereof, which may be optionally        be in the form of a hydrate, solvate, stereoisomer, or        polymorph, for use in treating a medical disorder that responds        to vascular remodeling effects and/or anti-CAF activity (e.g., a        proliferative disease such as cancer or a tumor);    -   (iv) A compound of Formula (I), (II), (III), or (IV), or a        pharmaceutically acceptable salt or isotopically labeled        derivative, which may be optionally be in the form of a hydrate,        solvate, stereoisomer, or polymorph, for use to treat a        proliferative disease. In certain embodiments, the proliferative        disease is cancer. In certain embodiments, the cancer is        squamous cell carcinoma of the head and neck (SCCHN), breast        cancer, esophageal cancer, uterine cancer, ovarian cancer,        colorectal cancer, endometrial cancer, or a sarcoma;    -   (v) A compound of Formula (I), (II), (III), or (IV), or a        pharmaceutically acceptable salt or isotopically labeled        derivative thereof, which may be optionally be in the form of a        hydrate, solvate, stereoisomer, or polymorph, for use in        treating a medical disorder that responds to vascular remodeling        effects and/or anti-CAF activity (e.g., a proliferative disease        such as a cancer or tumor);    -   (vi) A deuterated derivative of a compound of Formula (I), (II),        (III), or (IV), or a pharmaceutically acceptable salt thereof;    -   (vii) A process for manufacturing a medicament intended for the        therapeutic use for treating or preventing a disorder such as a        proliferative disease (e.g., cancer or tumor) that responds to        vascular remodeling effects and/or anti-CAF activity,        characterized in that a compound provided herein, or a        pharmaceutically acceptable salt or isotopically labeled        derivative thereof, which may be optionally be in the form of a        hydrate, solvate, stereoisomer, or polymorph described above, or        an embodiment of the active compound, is used in the        manufacture;    -   (viii) A compound of Formula (I), (II), (III), or (IV), or a        pharmaceutically acceptable salt or isotopically labeled        derivative thereof, in substantially pure form (e.g., at least        90 or 95%);    -   (ix) A pharmaceutically acceptable composition of a compound        provided herein, or a pharmaceutically acceptable salt or        isotopically labeled derivative thereof, which may be optionally        be in the form of a hydrate, solvate, stereoisomer, or        polymorph, in a pharmaceutically acceptable carrier or        excipient;    -   (x) A pharmaceutically acceptable dosage form of a compound        provided herein, or a pharmaceutically acceptable salt or        isotopically labeled derivative thereof, which may be optionally        be in the form of a hydrate, solvate, stereoisomer, or        polymorph, optionally in a pharmaceutically acceptable carrier        or excipient;    -   (xi) A compound of Formula (I), (II), (III), or (IV), or a        pharmaceutically acceptable salt or isotopically labeled        derivative thereof, to treat a disorder described herein (e.g.,        proliferative disease) whereby it acts through a mechanism other        than vascular remodeling effects and/or anti-CAF activity of        action; and    -   (xii) Methods for the manufacture of the compounds described        herein, and intermediates in the synthesis.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments of theinvention and together with the description, provide non-limitingexamples of the invention.

FIG. 1 shows antitumor effects of Compound (1) in FaDu subcutaneousxenograft model (head and neck cancer) in mice as monotherapy asdescribed in Pharmacological Test Example 4.

FIG. 2 shows antitumor activity of Compound (1) against OSC-19subcutaneous xenograft model (head and neck cancer) in mice asmonotherapy as described in Pharmacological Test Example 5.

FIG. 3 shows antitumor activity of Compound (1) against HCC-1806subcutaneous xenograft (breast cancer) model in mice as monotherapy asdescribed in Pharmacological Test Example 6.

FIG. 4 shows antitumor effects of Compound (1) in FaDu subcutaneousxenograft model in combination with cetuximab in mice as described inPharmacological Test Example 7.

FIG. 5 shows antitumor activity of Compound (1) in KPL-4 subcutaneousxenograft model (breast cancer) in combination with trastuzumab in miceas described in Pharmacological Test Example 8.

FIG. 6A-6B show anti-tumor effect of Compound (1) in HSC-2 orthotopictransplantation mouse model. FIG. 6A. Nude mice were implanted withluciferase-transduced HSC-2 (1×10⁶ cells/spot) in tongue. The amount ofluciferase-transduced HSC-2 was analyzed using In Vivo Imaging System(IVIS). Data show the bioluminescence levels in tongue in each mouse.FIG. 6B. Representative bioluminescence image of 16 mice. CDDP, CTX,CDDP+CTX were used for as comparators, which are currently used intreatment of SCCHN cancer patient treatment. CDDP=cisplatin,CTX=cetuximab.

FIG. 7A-7B show survival advantage of Compound (1) in combination withcetuximab in HSC-2 orthotopic transplantation mouse model. FIG. 7A. Nudemice were implanted with luciferase-transduced HSC-2 (1×10⁶ cells/spot)in tongue. Data show the survival curve until Day 100 after treatment ofdrugs (n=16). *P<0.0001 versus Compound (1) or CTX alone (Log-rank(Mantel-Cox) test). FIG. 7B. The amount of luciferase-transduced HSC-2was analyzed using In Vivo Imaging System (IVIS). Bioluminescence imagesof 10 survived mice of Compound (1)+CTX combination group on Day 100.RBW=relative body weight. CDDP=cisplatin, CTX=cetuximab.

FIG. 8A-8B show anti-tumor effect of Compound (1) in combination withradiation therapy in FaDu mouse xenograft model. FIG. 8A. Nude mice weresubcutaneously implanted with luciferase-transduced FaDu (5×10⁶cells/spot) in the right thighs. Thirteen days after the inoculation,mice were randomly assigned (n=6), and intravenously injected withCompound (1) at 90 μg/kg on Day 1 and Day 8 with or without RT of 18 Gyon Day 4 and Day 11. The amount of luciferase-transduced FaDu wasanalyzed using In Vivo Imaging System (IVIS). Data show the meanrelative bioluminescence level to Day 1 and SEM (n=6). SEM=standarderror of the mean. * P<0.05 versus non-treated on Day 29 (unpairedt-test). FIG. 8B. Representative bioluminescence images of 6 mice eachgroup on Day 29. RT=radiation therapy.

FIG. 9 shows anti-tumor activities of Compound (1) in combination withanti-mPD-1 antibody. CT26 s.c. syngeneic mouse model (colon carcinoma)was treated with Compound (1) and anti-mPD-1 antibody in Q7D scheduleand twice a week schedule, respectively, for 3 weeks. Results showmeans±SEM of tumor volumes (mm³) (n=8).

FIG. 10A shows a cell-free tubulin polymerization assay. Compound (1)has inhibitory activity on tubulin polymerization. FIG. 10B shows amicrotubule dynamics assay. Compound (1) also has inhibitory activity onmicrotubule dynamics.

FIG. 11 shows that Compound (1) is a potent antiproliferative agent inesophageal cancer (OE21, OE33, and TE-8) and uterine cancer (MES-SA,MES-SA/Dx5-Rx1) cell lines.

FIG. 12 shows that Compound (1) has potent anti-tumor activity insubcutaneous xenograft models of breast and ovarian cancer (KPL-4 andCOLO-704, respectively) as a monotherapy.

FIG. 13 shows the effect of Compound (1) on tumor microenvironments. Asshown, Compound (1) increases microvessel density. * P<0.05, ** P<0.01,****P<0.0001 versus non-treat (Dunnett multiple comparison test).

FIG. 14 shows the effect of Compound (1) on tumor microenvironments. Asshown, Compound (1) reduces α-SMA positive CAFs.

FIG. 15 shows that Compound (1) decreases ECM proteins from CAFs in FaDusubcutaneous xenograft model. FaDu xenograft tumors were collected onDay 6 after single administration of Compound (1) 180 μg/kg+cetuximab onDay 1.

FIG. 16 shows that Compound (1) exhibits a dose-dependent combinationaleffect with cetuximab in a FaDu subcutaneous xenograft model. Singledose, n=6. Compound (1) and cetuximab (CTX) were administered on Day 1in the FaDu xenograft model.

FIG. 17 shows antitumor effects in the soft tissue sarcoma xenograftmodels in mice as monotherapy. MES-SA (human uterine sarcoma), HT-1080(human fibrosarcoma), and CTG-2041 (human angiosarcoma) are shown.

FIG. 18 shows antitumor effects in endometrial cancer xenograft modelsin mice as monotherapy. HEC-108 and AN3CA (endometrial cancer) areshown.

FIGS. 19A-19H show that Compound (1) reduced TGF-β-induced α-SMAexpression in an in vitro CAF-inducing system. FIG. 19A. BJ cells(normal human lung fibroblasts) were co-cultured with FaDu cells forthree days in the absence (vehicle) or presence of A83-01, a potentselective TGF-β-R inhibitor, and the expression of α-SMA was analysed byimmunofluorescence staining (red color). Samples were also stained withanti-pan-human cytokeratin (green color) for cancer cell staining andDAPI (blue color) for nuclear staining. FIGS. 19B-19E. BJ cells weretreated with Compound (1) (0.15 nmol/L in immunofluorescence stainingdata and indicated concentrations in western blot analysis data) andTGF-β for 2 days. FIGS. 19B-19E. Samples were stained with the indicatedantibody (red color) and DAPI (blue color) for nuclear staining. FIG.19E Western blot images and quantification of the images. The graphshows ratios of the results of treatment groups to the non-treatedgroup. FIGS. 19G and 19E. BJ cells were pretreated with defactinib (1μmol/L) and TGF-β for 2 days. The lens magnification used was ×4 (FIGS.19A, 19B, 19C, 19G, and 19H) or ×40 (FIGS. 19D and 19E).

FIGS. 20A and 20B. Quantification of immunofluorescence images of BJcells treated with TGF-β (1 ng/mL) and the indicated concentrations ofCompound (1) for α-SMA (FIG. 20A), Phospho-S6-ribosomal protein(Ser235/236) (FIG. 20B). FIG. 20C. Immunofluorescence analysis of BJcells treated with TGF-β in the absence or presence of dactolisib, aphosphatidylinositol 3 kinase inhibitor. FIG. 20D. Immunofluorescenceanalysis of phosphorylated Smad2/3 in BJ cells pretreated with Compound(1) (72 hours) after 30 minutes of stimulation with TGF-β.Acetylated-α-tubulin was co-stained to confirm the activity of Compound(1). FIG. 20E. Western blot analysis of Smad2/3 in BJ cells treated withTGF-β and the indicated concentrations of Compound (1). The lensmagnification used was ×4 (FIG. 20C) or ×40 (FIG. 20D).

FIG. 21A. Immunofluorescence staining of α-SMA (red color) andpan-cytokeratin (green color) in TIG3 co-cultured with FaDu in theabsence or presence of A83-01, an FAK inhibitor. FIGS. 21B and 21C.Quantification of immunofluorescence images of TIG3 cells treated withTGF-β and the indicated concentrations of Compound (1) for α-SMA (FIG.21B), Phospho-S6-ribosomal protein (Ser235/236) (FIG. 21C). FIGS. 21Dand 21E. Immunofluorescence analysis of TIG3 cells treated with TGF-β inthe absence or presence of Compound (1) for β-tubulin (FIG. 21D),Phospho-FAK (Tyr397) (FIG. 21E). FIG. 21E Immunofluorescence analysis ofTIG3 cells treated with TGF-β in the absence or presence of dactolisib100 nmol/L for α-SMA. FIGS. 21G and 21H Immunofluorescence analysis ofTIG3 cells treated with TGF-β in the absence or presence of defactinib 3μmol/L for α-SMA (FIG. 21G), Phospho-S6-ribosomal protein (Ser235/236)(FIG. 21H). The lens magnification was ×4 (FIGS. 21A, 21F-21H) or ×40(FIGS. 21D and 21E).

FIG. 22 . Antitumor effect of Compound (1) in an HS-SY-II (humansynovial sarcoma) xenograft model in mice as a monotherapy.

FIG. 23 . Antitumor effect of Compound (1) in an HuTu 80 (human duodenalcell) xenograft model in mice as a monotherapy.

DEFINITIONS Chemical Definitions

Definitions of specific functional groups and chemical terms aredescribed in more detail below. The chemical elements are identified inaccordance with the Periodic Table of the Elements, CAS version,Handbook of Chemistry and Physics, 75^(th) Ed., inside cover, andspecific functional groups are generally defined as described therein.Additionally, general principles of organic chemistry, as well asspecific functional moieties and reactivity, are described in OrganicChemistry, Thomas Sorrell, University Science Books, Sausalito, 1999;Smith and March, March s Advanced Organic Chemistry, 5^(th) Edition,John Wiley & Sons, Inc., New York, 2001; Larock, Comprehensive OrganicTransformations, VCH Publishers, Inc., New York, 1989; and Carruthers,Some Modern Methods of Organic Synthesis, 3^(rd) Edition, CambridgeUniversity Press, Cambridge, 1987.

Compounds described herein can comprise one or more asymmetric centers,and thus can exist in various stereoisomeric forms, e.g., enantiomersand/or diastereomers. For example, the compounds described herein can bein the form of an individual enantiomer, diastereomer or geometricisomer, or can be in the form of a mixture of stereoisomers, includingracemic mixtures and mixtures enriched in one or more stereoisomer.Isomers can be isolated from mixtures by methods known to those skilledin the art, including chiral high pressure liquid chromatography (HPLC)and the formation and crystallization of chiral salts; or preferredisomers can be prepared by asymmetric syntheses. See, for example,Jacques et al., Enantiomers, Racemates and Resolutions (WileyInterscience, New York, 1981); Wilen et al., Tetrahedron 33:2725 (1977);Eliel, E. L. Stereochemistry of Carbon Compounds (McGraw-Hill, N Y,1962); and Wilen, S. H., Tables of Resolving Agents and OpticalResolutions p. 268 (E.L. Eliel, Ed., Univ. of Notre Dame Press, NotreDame, Ind. 1972). The invention additionally encompasses compounds asindividual isomers substantially free of other isomers, andalternatively, as mixtures of various isomers.

Unless otherwise stated, structures depicted herein are also meant toinclude compounds that differ only in the presence of one or moreisotopically enriched atoms. For example, compounds having the presentstructures except for the replacement of hydrogen by deuterium ortritium, replacement of ¹⁹F with ¹⁸F, or the replacement of ¹²C with ¹³Cor ¹⁴C are within the scope of the disclosure. Such compounds areuseful, for example, as analytical tools or probes in biological assays.

When a range of values is listed, it is intended to encompass each valueand sub-range within the range. For example, “C₁₋₆ alkyl” is intended toencompass, C₁, C₂, C₃, C₄, C₅, C₆, C₁₋₆, C₁₋₅, C₁₋₄, C₁₋₃, C₁₋₂, C₂₋₆,C₂₋₅, C₂₋₄, C₂₋₃, C₃₋₆, C₃₋₅, C₃₋₄, C₄₋₆, C₄₋₅, and C₅₋₆ alkyl.

The term “aliphatic” refers to alkyl, alkenyl, alkynyl, and carbocyclicgroups. Likewise, the term “heteroaliphatic” refers to heteroalkyl,heteroalkenyl, heteroalkynyl, and heterocyclic groups.

The term “alkyl” refers to a radical of a straight-chain or branchedsaturated hydrocarbon group having from 1 to 10 carbon atoms (“C₁₋₁₀alkyl”). In some embodiments, an alkyl group has 1 to 9 carbon atoms(“C₁₋₉ alkyl”). In some embodiments, an alkyl group has 1 to 8 carbonatoms (“C₁₋₈ alkyl”). In some embodiments, an alkyl group has 1 to 7carbon atoms (“C₁₋₇ alkyl”). In some embodiments, an alkyl group has 1to 6 carbon atoms (“C₁₋₆ alkyl”). In some embodiments, an alkyl grouphas 1 to 5 carbon atoms (“C₁₋₅ alkyl”). In some embodiments, an alkylgroup has 1 to 4 carbon atoms (“C₁₋₄ alkyl”). In some embodiments, analkyl group has 1 to 3 carbon atoms (“C₁₋₃ alkyl”). In some embodiments,an alkyl group has 1 to 2 carbon atoms (“C₁₋₂ alkyl”). In someembodiments, an alkyl group has 1 carbon atom (“C₁ alkyl”). In someembodiments, an alkyl group has 2 to 6 carbon atoms (“C₂₋₆ alkyl”).Examples of C₁₋₆ alkyl groups include methyl (C₁), ethyl (C₂), propyl(C₃) (e.g., n-propyl, iso-propyl), butyl (C₄) (e.g., n-butyl,tert-butyl, sec-butyl, iso-butyl), pentyl (C₅) (e.g., n-pentyl,3-pentanyl, amyl, neopentyl, 3-methyl-2-butanyl, tertiary amyl), andhexyl (C₆) (e.g., n-hexyl). Additional examples of alkyl groups includen-heptyl (C₇), n-octyl (C₈), and the like. Unless otherwise specified,each instance of an alkyl group is independently unsubstituted (an“unsubstituted alkyl”) or substituted (a “substituted alkyl”) with oneor more substituents (e.g., halogen, such as F). In certain embodiments,the alkyl group is an unsubstituted C₁₋₁₀ alkyl (such as unsubstitutedC₁₋₆ alkyl, e.g., —CH₃ (Me), unsubstituted ethyl (Et), unsubstitutedpropyl (Pr, e.g., unsubstituted n-propyl (n-Pr), unsubstitutediso-propyl (i-Pr)), unsubstituted butyl (Bu, e.g., unsubstituted n-butyl(n-Bu), unsubstituted tert-butyl (tert-Bu or t-Bu), unsubstitutedsec-butyl (sec-Bu), unsubstituted iso-butyl (i-Bu)). In certainembodiments, the alkyl group is a substituted C₁₋₁₀ alkyl (such assubstituted C₁₋₆ alkyl, e.g., —CF₃, Bn).

The term “haloalkyl” is a substituted alkyl group, wherein one or moreof the hydrogen atoms are independently replaced by a halogen, e.g.,fluoro, bromo, chloro, or iodo. In some embodiments, the haloalkylmoiety has 1 to 8 carbon atoms (“C₁₋₈ haloalkyl”). In some embodiments,the haloalkyl moiety has 1 to 6 carbon atoms (“C₁₋₆ haloalkyl”). In someembodiments, the haloalkyl moiety has 1 to 4 carbon atoms (“C¹⁻⁴haloalkyl”). In some embodiments, the haloalkyl moiety has 1 to 3 carbonatoms (“C₁₋₃ haloalkyl”). In some embodiments, the haloalkyl moiety has1 to 2 carbon atoms (“C₁₋₂ haloalkyl”). Examples of haloalkyl groupsinclude —CHF₂, —CH₂F, —CF₃, —CH₂CF₃, —CF₂CF₃, —CF₂CF₂CF₃, —CCl₃, —CFCl₂,—CF₂Cl, and the like.

The term “heteroalkyl” refers to an alkyl group, which further includesat least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms) selected fromoxygen, nitrogen, or sulfur within (i.e., inserted between adjacentcarbon atoms of) and/or placed at one or more terminal position(s) ofthe parent chain. In certain embodiments, a heteroalkyl group refers toa saturated group having from 1 to 10 carbon atoms and 1 or moreheteroatoms within the parent chain (“heteroC₁₋₁₀ alkyl”). In someembodiments, a heteroalkyl group is a saturated group having 1 to 9carbon atoms and 1 or more heteroatoms within the parent chain(“heteroC₁₋₉ alkyl”). In some embodiments, a heteroalkyl group is asaturated group having 1 to 8 carbon atoms and 1 or more heteroatomswithin the parent chain (“heteroC₁₋₈ alkyl”). In some embodiments, aheteroalkyl group is a saturated group having 1 to 7 carbon atoms and 1or more heteroatoms within the parent chain (“heteroC₁₋₇ alkyl”). Insome embodiments, a heteroalkyl group is a saturated group having 1 to 6carbon atoms and 1 or more heteroatoms within the parent chain(“heteroC₁₋₆ alkyl”). In some embodiments, a heteroalkyl group is asaturated group having 1 to 5 carbon atoms and 1 or 2 heteroatoms withinthe parent chain (“heteroC₁₋₅ alkyl”). In some embodiments, aheteroalkyl group is a saturated group having 1 to 4 carbon atoms and 1or 2 heteroatoms within the parent chain (“heteroC₁₋₄ alkyl”). In someembodiments, a heteroalkyl group is a saturated group having 1 to 3carbon atoms and 1 heteroatom within the parent chain (“heteroC₁₋₃alkyl”). In some embodiments, a heteroalkyl group is a saturated grouphaving 1 to 2 carbon atoms and 1 heteroatom within the parent chain(“heteroC₁₋₂ alkyl”). In some embodiments, a heteroalkyl group is asaturated group having 1 carbon atom and 1 heteroatom (“heteroC₁alkyl”). In some embodiments, a heteroalkyl group is a saturated grouphaving 2 to 6 carbon atoms and 1 or 2 heteroatoms within the parentchain (“heteroC₂₋₆ alkyl”). Unless otherwise specified, each instance ofa heteroalkyl group is independently unsubstituted (an “unsubstitutedheteroalkyl”) or substituted (a “substituted heteroalkyl”) with one ormore substituents. In certain embodiments, the heteroalkyl group is anunsubstituted heteroC₁₋₁₀ alkyl. In certain embodiments, the heteroalkylgroup is a substituted heteroC₁₋₁₀ alkyl.

The term “alkenyl” refers to a radical of a straight-chain or branchedhydrocarbon group having from 2 to 10 carbon atoms and one or morecarbon-carbon double bonds (e.g., 1, 2, 3, or 4 double bonds). In someembodiments, an alkenyl group has 2 to 9 carbon atoms (“C₂₋₉ alkenyl”).In some embodiments, an alkenyl group has 2 to 8 carbon atoms (“C₂₋₈alkenyl”). In some embodiments, an alkenyl group has 2 to 7 carbon atoms(“C₂₋₇ alkenyl”). In some embodiments, an alkenyl group has 2 to 6carbon atoms (“C₂₋₆ alkenyl”). In some embodiments, an alkenyl group has2 to 5 carbon atoms (“C₂₋₅ alkenyl”). In some embodiments, an alkenylgroup has 2 to 4 carbon atoms (“C₂₋₄ alkenyl”). In some embodiments, analkenyl group has 2 to 3 carbon atoms (“C₂₋₃ alkenyl”). In someembodiments, an alkenyl group has 2 carbon atoms (“C₂ alkenyl”). The oneor more carbon-carbon double bonds can be internal (such as in2-butenyl) or terminal (such as in 1-butenyl). Examples of C₂₋₄ alkenylgroups include ethenyl (C₂), 1-propenyl (C₃), 2-propenyl (C₃), 1-butenyl(C₄), 2-butenyl (C₄), butadienyl (C₄), and the like. Examples of C₂₋₆alkenyl groups include the aforementioned C₂₋₄ alkenyl groups as well aspentenyl (C₅), pentadienyl (C₅), hexenyl (C₆), and the like. Additionalexamples of alkenyl include heptenyl (C₇), octenyl (C₈), octatrienyl(C₈), and the like. Unless otherwise specified, each instance of analkenyl group is independently unsubstituted (an “unsubstitutedalkenyl”) or substituted (a “substituted alkenyl”) with one or moresubstituents. In certain embodiments, the alkenyl group is anunsubstituted C₂₋₁₀ alkenyl. In certain embodiments, the alkenyl groupis a substituted C₂₋₁₀ alkenyl. In an alkenyl group, a C═C double bondfor which the stereochemistry is not specified (e.g., —CH═CHCH₃ or

may be an (E)- or (Z)-double bond.

The term “heteroalkenyl” refers to an alkenyl group, which furtherincludes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms)selected from oxygen, nitrogen, or sulfur within (i.e., inserted betweenadjacent carbon atoms of) and/or placed at one or more terminalposition(s) of the parent chain. In certain embodiments, a heteroalkenylgroup refers to a group having from 2 to 10 carbon atoms, at least onedouble bond, and 1 or more heteroatoms within the parent chain(“heteroC₂₋₁₀ alkenyl”). In some embodiments, a heteroalkenyl group has2 to 9 carbon atoms at least one double bond, and 1 or more heteroatomswithin the parent chain (“heteroC₂₋₉ alkenyl”). In some embodiments, aheteroalkenyl group has 2 to 8 carbon atoms, at least one double bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₈alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 7 carbonatoms, at least one double bond, and 1 or more heteroatoms within theparent chain (“heteroC₂₋₇ alkenyl”). In some embodiments, aheteroalkenyl group has 2 to 6 carbon atoms, at least one double bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₆alkenyl”). In some embodiments, a heteroalkenyl group has 2 to 5 carbonatoms, at least one double bond, and 1 or 2 heteroatoms within theparent chain (“heteroC₂₋₅ alkenyl”). In some embodiments, aheteroalkenyl group has 2 to 4 carbon atoms, at least one double bond,and 1 or 2 heteroatoms within the parent chain (“heteroC₂₋₄ alkenyl”).In some embodiments, a heteroalkenyl group has 2 to 3 carbon atoms, atleast one double bond, and 1 heteroatom within the parent chain(“heteroC₂₋₃ alkenyl”). In some embodiments, a heteroalkenyl group has 2to 6 carbon atoms, at least one double bond, and 1 or 2 heteroatomswithin the parent chain (“heteroC₂₋₆ alkenyl”). Unless otherwisespecified, each instance of a heteroalkenyl group is independentlyunsubstituted (an “unsubstituted heteroalkenyl”) or substituted (a“substituted heteroalkenyl”) with one or more substituents. In certainembodiments, the heteroalkenyl group is an unsubstituted heteroC₂₋₁₀alkenyl. In certain embodiments, the heteroalkenyl group is asubstituted heteroC₂₋₁₀ alkenyl.

The term “alkynyl” refers to a radical of a straight-chain or branchedhydrocarbon group having from 2 to 10 carbon atoms and one or morecarbon-carbon triple bonds (e.g., 1, 2, 3, or 4 triple bonds) (“C₂₋₁₀alkynyl”). In some embodiments, an alkynyl group has 2 to 9 carbon atoms(“C₂₋₉ alkynyl”). In some embodiments, an alkynyl group has 2 to 8carbon atoms (“C₂₋₈ alkynyl”). In some embodiments, an alkynyl group has2 to 7 carbon atoms (“C₂₋₇ alkynyl”). In some embodiments, an alkynylgroup has 2 to 6 carbon atoms (“C₂₋₆ alkynyl”). In some embodiments, analkynyl group has 2 to 5 carbon atoms (“C₂₋₅ alkynyl”). In someembodiments, an alkynyl group has 2 to 4 carbon atoms (“C₂₋₄ alkynyl”).In some embodiments, an alkynyl group has 2 to 3 carbon atoms (“C₂₋₃alkynyl”). In some embodiments, an alkynyl group has 2 carbon atoms (“C₂alkynyl”). The one or more carbon-carbon triple bonds can be internal(such as in 2-butynyl) or terminal (such as in 1-butynyl). Examples ofC₂₋₄ alkynyl groups include, without limitation, ethynyl (C₂),1-propynyl (C₃), 2-propynyl (C₃), 1-butynyl (C₄), 2-butynyl (C₄), andthe like. Examples of C₂₋₆ alkenyl groups include the aforementionedC₂₋₄ alkynyl groups as well as pentynyl (C₈), hexynyl (C₆), and thelike. Additional examples of alkynyl include heptynyl (C₇), octynyl(C₈), and the like. Unless otherwise specified, each instance of analkynyl group is independently unsubstituted (an “unsubstitutedalkynyl”) or substituted (a “substituted alkynyl”) with one or moresubstituents. In certain embodiments, the alkynyl group is anunsubstituted C₂₋₁₀ alkynyl. In certain embodiments, the alkynyl groupis a substituted C₂₋₁₀ alkynyl.

The term “heteroalkynyl” refers to an alkynyl group, which furtherincludes at least one heteroatom (e.g., 1, 2, 3, or 4 heteroatoms)selected from oxygen, nitrogen, or sulfur within (i.e., inserted betweenadjacent carbon atoms of) and/or placed at one or more terminalposition(s) of the parent chain. In certain embodiments, a heteroalkynylgroup refers to a group having from 2 to 10 carbon atoms, at least onetriple bond, and 1 or more heteroatoms within the parent chain(“heteroC₂₋₁₀ alkynyl”). In some embodiments, a heteroalkynyl group has2 to 9 carbon atoms, at least one triple bond, and 1 or more heteroatomswithin the parent chain (“heteroC₂₋₉ alkynyl”). In some embodiments, aheteroalkynyl group has 2 to 8 carbon atoms, at least one triple bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₈alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 7 carbonatoms, at least one triple bond, and 1 or more heteroatoms within theparent chain (“heteroC₂₋₇ alkynyl”). In some embodiments, aheteroalkynyl group has 2 to 6 carbon atoms, at least one triple bond,and 1 or more heteroatoms within the parent chain (“heteroC₂₋₆alkynyl”). In some embodiments, a heteroalkynyl group has 2 to 5 carbonatoms, at least one triple bond, and 1 or 2 heteroatoms within theparent chain (“heteroC₂₋₅ alkynyl”). In some embodiments, aheteroalkynyl group has 2 to 4 carbon atoms, at least one triple bond,and 1 or 2 heteroatoms within the parent chain (“heteroC₂₋₄ alkynyl”).In some embodiments, a heteroalkynyl group has 2 to 3 carbon atoms, atleast one triple bond, and 1 heteroatom within the parent chain(“heteroC₂₋₃ alkynyl”). In some embodiments, a heteroalkynyl group has 2to 6 carbon atoms, at least one triple bond, and 1 or 2 heteroatomswithin the parent chain (“heteroC₂₋₆ alkynyl”).

Unless otherwise specified, each instance of a heteroalkynyl group isindependently unsubstituted (an “unsubstituted heteroalkynyl”) orsubstituted (a “substituted heteroalkynyl”) with one or moresubstituents. In certain embodiments, the heteroalkynyl group is anunsubstituted heteroC₂₋₁₀ alkynyl. In certain embodiments, theheteroalkynyl group is a substituted heteroC₂₋₁₀ alkynyl.

The term “carbocyclyl” or “carbocyclic” refers to a radical of anon-aromatic cyclic hydrocarbon group having from 3 to 14 ring carbonatoms (“C₃₋₁₄ carbocyclyl”) and zero heteroatoms in the non-aromaticring system. In some embodiments, a carbocyclyl group has 3 to 10 ringcarbon atoms (“C₃₋₁₀ carbocyclyl”). In some embodiments, a carbocyclylgroup has 3 to 8 ring carbon atoms (“C₃₋₈ carbocyclyl”). In someembodiments, a carbocyclyl group has 3 to 7 ring carbon atoms (“C₃₋₇carbocyclyl”). In some embodiments, a carbocyclyl group has 3 to 6 ringcarbon atoms (“C₃₋₆ carbocyclyl”). In some embodiments, a carbocyclylgroup has 4 to 6 ring carbon atoms (“C₄₋₆ carbocyclyl”). In someembodiments, a carbocyclyl group has 5 to 6 ring carbon atoms (“C₅₋₆carbocyclyl”). In some embodiments, a carbocyclyl group has 5 to 10 ringcarbon atoms (“C₅₋₁₀ carbocyclyl”). Exemplary C₃₋₆ carbocyclyl groupsinclude, without limitation, cyclopropyl (C₃), cyclopropenyl (C₃),cyclobutyl (C₄), cyclobutenyl (C₄), cyclopentyl (C₅), cyclopentenyl(C₅), cyclohexyl (C₆), cyclohexenyl (C₆), cyclohexadienyl (C₆), and thelike. Exemplary C₃₋₈ carbocyclyl groups include, without limitation, theaforementioned C₃₋₆ carbocyclyl groups as well as cycloheptyl (C₇),cycloheptenyl (C₇), cycloheptadienyl (C₇), cycloheptatrienyl (C₇),cyclooctyl (C₈), cyclooctenyl (C₈), bicyclo[2.2.1]heptanyl (C₇),bicyclo[2.2.2]octanyl (C₈), and the like. Exemplary C₃₋₁₀ carbocyclylgroups include, without limitation, the aforementioned C₃₋₈ carbocyclylgroups as well as cyclononyl (C₉), cyclononenyl (C₉), cyclodecyl (C₁₀),cyclodecenyl (C₁₀), octahydro-1H-indenyl (C₉), decahydronaphthalenyl(C₁₀), spiro[4.5]decanyl (C₁₀), and the like. As the foregoing examplesillustrate, in certain embodiments, the carbocyclyl group is eithermonocyclic (“monocyclic carbocyclyl”) or polycyclic (e.g., containing afused, bridged or spiro ring system such as a bicyclic system (“bicycliccarbocyclyl”) or tricyclic system (“tricyclic carbocyclyl”)) and can besaturated or can contain one or more carbon-carbon double or triplebonds. “Carbocyclyl” also includes ring systems wherein the carbocyclylring, as defined above, is fused with one or more aryl or heteroarylgroups wherein the point of attachment is on the carbocyclyl ring, andin such instances, the number of carbons continue to designate thenumber of carbons in the carbocyclic ring system. Unless otherwisespecified, each instance of a carbocyclyl group is independentlyunsubstituted (an “unsubstituted carbocyclyl”) or substituted (a“substituted carbocyclyl”) with one or more substituents. In certainembodiments, the carbocyclyl group is an unsubstituted C₃₋₁₄carbocyclyl. In certain embodiments, the carbocyclyl group is asubstituted C₃₋₁₄ carbocyclyl.

In some embodiments, “carbocyclyl” is a monocyclic, saturatedcarbocyclyl group having from 3 to 14 ring carbon atoms (“C₃₋₁₄cycloalkyl”). In some embodiments, a cycloalkyl group has 3 to 10 ringcarbon atoms (“C₃₋₁₀ cycloalkyl”). In some embodiments, a cycloalkylgroup has 3 to 8 ring carbon atoms (“C₃₋₈ cycloalkyl”). In someembodiments, a cycloalkyl group has 3 to 6 ring carbon atoms (“C₃₋₆cycloalkyl”). In some embodiments, a cycloalkyl group has 4 to 6 ringcarbon atoms (“C₄₋₆ cycloalkyl”). In some embodiments, a cycloalkylgroup has 5 to 6 ring carbon atoms (“C₅₋₆ cycloalkyl”). In someembodiments, a cycloalkyl group has 5 to 10 ring carbon atoms (“C₅₋₁₀cycloalkyl”). Examples of C₅₋₆ cycloalkyl groups include cyclopentyl(C₅) and cyclohexyl (C₅). Examples of C₃₋₆ cycloalkyl groups include theaforementioned C₅₋₆ cycloalkyl groups as well as cyclopropyl (C₃) andcyclobutyl (C₄). Examples of C₃₋₈ cycloalkyl groups include theaforementioned C₃₋₆ cycloalkyl groups as well as cycloheptyl (C₇) andcyclooctyl (C₈). Unless otherwise specified, each instance of acycloalkyl group is independently unsubstituted (an “unsubstitutedcycloalkyl”) or substituted (a “substituted cycloalkyl”) with one ormore substituents. In certain embodiments, the cycloalkyl group is anunsubstituted C₃₋₁₄ cycloalkyl. In certain embodiments, the cycloalkylgroup is a substituted C₃₋₁₄ cycloalkyl.

The term “heterocyclyl” or “heterocyclic” refers to a radical of a 3- to14-membered non-aromatic ring system having ring carbon atoms and 1 to 4ring heteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“3-14 membered heterocyclyl”). Inheterocyclyl groups that contain one or more nitrogen atoms, the pointof attachment can be a carbon or nitrogen atom, as valency permits. Aheterocyclyl group can either be monocyclic (“monocyclic heterocyclyl”)or polycyclic (e.g., a fused, bridged or spiro ring system such as abicyclic system (“bicyclic heterocyclyl”) or tricyclic system(“tricyclic heterocyclyl”)), and can be saturated or can contain one ormore carbon-carbon double or triple bonds. Heterocyclyl polycyclic ringsystems can include one or more heteroatoms in one or both rings.“Heterocyclyl” also includes ring systems wherein the heterocyclyl ring,as defined above, is fused with one or more carbocyclyl groups whereinthe point of attachment is either on the carbocyclyl or heterocyclylring, or ring systems wherein the heterocyclyl ring, as defined above,is fused with one or more aryl or heteroaryl groups, wherein the pointof attachment is on the heterocyclyl ring, and in such instances, thenumber of ring members continue to designate the number of ring membersin the heterocyclyl ring system. Unless otherwise specified, eachinstance of heterocyclyl is independently unsubstituted (an“unsubstituted heterocyclyl”) or substituted (a “substitutedheterocyclyl”) with one or more substituents. In certain embodiments,the heterocyclyl group is an unsubstituted 3-14 membered heterocyclyl.In certain embodiments, the heterocyclyl group is a substituted 3-14membered heterocyclyl.

In some embodiments, a heterocyclyl group is a 5-10 memberednon-aromatic ring system having ring carbon atoms and 1-4 ringheteroatoms, wherein each heteroatom is independently selected fromnitrogen, oxygen, and sulfur (“5-10 membered heterocyclyl”). In someembodiments, a heterocyclyl group is a 5-8 membered non-aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms, wherein eachheteroatom is independently selected from nitrogen, oxygen, and sulfur(“5-8 membered heterocyclyl”). In some embodiments, a heterocyclyl groupis a 5-6 membered non-aromatic ring system having ring carbon atoms and1-4 ring heteroatoms, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-6 membered heterocyclyl”). In someembodiments, the 5-6 membered heterocyclyl has 1-3 ring heteroatomsselected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6membered heterocyclyl has 1-2 ring heteroatoms selected from nitrogen,oxygen, and sulfur. In some embodiments, the 5-6 membered heterocyclylhas 1 ring heteroatom selected from nitrogen, oxygen, and sulfur.

Exemplary 3-membered heterocyclyl groups containing 1 heteroatominclude, without limitation, azirdinyl, oxiranyl, and thiiranyl.Exemplary 4-membered heterocyclyl groups containing 1 heteroatominclude, without limitation, azetidinyl, oxetanyl, and thietanyl.Exemplary 5-membered heterocyclyl groups containing 1 heteroatominclude, without limitation, tetrahydrofuranyl, dihydrofuranyl,tetrahydrothiophenyl, dihydrothiophenyl, pyrrolidinyl, dihydropyrrolyl,and pyrrolyl-2,5-dione. Exemplary 5-membered heterocyclyl groupscontaining 2 heteroatoms include, without limitation, dioxolanyl,oxathiolanyl and dithiolanyl. Exemplary 5-membered heterocyclyl groupscontaining 3 heteroatoms include, without limitation, triazolinyl,oxadiazolinyl, and thiadiazolinyl. Exemplary 6-membered heterocyclylgroups containing 1 heteroatom include, without limitation, piperidinyl,tetrahydropyranyl, dihydropyridinyl, and thianyl. Exemplary 6-memberedheterocyclyl groups containing 2 heteroatoms include, withoutlimitation, piperazinyl, morpholinyl, dithianyl, and dioxanyl. Exemplary6-membered heterocyclyl groups containing 3 heteroatoms include, withoutlimitation, triazinyl. Exemplary 7-membered heterocyclyl groupscontaining 1 heteroatom include, without limitation, azepanyl, oxepanyland thiepanyl. Exemplary 8-membered heterocyclyl groups containing 1heteroatom include, without limitation, azocanyl, oxecanyl andthiocanyl. Exemplary bicyclic heterocyclyl groups include, withoutlimitation, indolinyl, isoindolinyl, dihydrobenzofuranyl,dihydrobenzothienyl, tetrahydrobenzothienyl, tetrahydrobenzofuranyl,tetrahydroindolyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl,decahydroquinolinyl, decahydroisoquinolinyl, octahydrochromenyl,octahydroisochromenyl, decahydronaphthyridinyl,decahydro-1,8-naphthyridinyl, octahydropyrrolo[3,2-b]pyrrole, indolinyl,phthalimidyl, naphthalimidyl, chromanyl, chromenyl,1H-benzo[e][1,4]diazepinyl, 1,4,5,7-tetrahydropyrano[3,4-b]pyrrolyl,5,6-dihydro-4H-furo[3,2-b]pyrrolyl, 6,7-dihydro-5H-furo[3,2-b]pyranyl,5,7-dihydro-4H-thieno[2,3-c]pyranyl,2,3-dihydro-1H-pyrrolo[2,3-b]pyridinyl, 2,3-dihydrofuro[2,3-b]pyridinyl,4,5,6,7-tetrahydro-1H-pyrrolo[2,3-b]pyridinyl,4,5,6,7-tetrahydrofuro[3,2-c]pyridinyl,4,5,6,7-tetrahydrothieno[3,2-b]pyridinyl,1,2,3,4-tetrahydro-1,6-naphthyridinyl, and the like.

The term “aryl” refers to a radical of a monocyclic or polycyclic (e.g.,bicyclic or tricyclic) 4n+2 aromatic ring system (e.g., having 6, 10, or14 π electrons shared in a cyclic array) having 6-14 ring carbon atomsand zero heteroatoms provided in the aromatic ring system (“C₆₋₁₄aryl”). In some embodiments, an aryl group has 6 ring carbon atoms (“C₆aryl”; e.g., phenyl). In some embodiments, an aryl group has 10 ringcarbon atoms (“C₁₀ aryl”; e.g., naphthyl such as 1-naphthyl and2-naphthyl). In some embodiments, an aryl group has 14 ring carbon atoms(“C₁₄ aryl”; e.g., anthracyl). “Aryl” also includes ring systems whereinthe aryl ring, as defined above, is fused with one or more carbocyclylor heterocyclyl groups wherein the radical or point of attachment is onthe aryl ring, and in such instances, the number of carbon atomscontinue to designate the number of carbon atoms in the aryl ringsystem. Unless otherwise specified, each instance of an aryl group isindependently unsubstituted (an “unsubstituted aryl”) or substituted (a“substituted aryl”) with one or more substituents. In certainembodiments, the aryl group is an unsubstituted C₆₋₁₄ aryl. In certainembodiments, the aryl group is a substituted C₆₋₁₄ aryl.

The term “heteroaryl” refers to a radical of a 5-14 membered monocyclicor polycyclic (e.g., bicyclic, tricyclic) 4n+2 aromatic ring system(e.g., having 6, 10, or 14 π electrons shared in a cyclic array) havingring carbon atoms and 1-4 ring heteroatoms provided in the aromatic ringsystem, wherein each heteroatom is independently selected from nitrogen,oxygen, and sulfur (“5-14 membered heteroaryl”). In heteroaryl groupsthat contain one or more nitrogen atoms, the point of attachment can bea carbon or nitrogen atom, as valency permits. Heteroaryl polycyclicring systems can include one or more heteroatoms in one or both rings.“Heteroaryl” includes ring systems wherein the heteroaryl ring, asdefined above, is fused with one or more carbocyclyl or heterocyclylgroups wherein the point of attachment is on the heteroaryl ring, and insuch instances, the number of ring members continue to designate thenumber of ring members in the heteroaryl ring system. “Heteroaryl” alsoincludes ring systems wherein the heteroaryl ring, as defined above, isfused with one or more aryl groups wherein the point of attachment iseither on the aryl or heteroaryl ring, and in such instances, the numberof ring members designates the number of ring members in the fusedpolycyclic (aryl/heteroaryl) ring system. Polycyclic heteroaryl groupswherein one ring does not contain a heteroatom (e.g., indolyl,quinolinyl, carbazolyl, and the like) the point of attachment can be oneither ring, i.e., either the ring bearing a heteroatom (e.g.,2-indolyl) or the ring that does not contain a heteroatom (e.g.,5-indolyl).

In some embodiments, a heteroaryl group is a 5-10 membered aromatic ringsystem having ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-10 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-8 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-8 membered heteroaryl”). In someembodiments, a heteroaryl group is a 5-6 membered aromatic ring systemhaving ring carbon atoms and 1-4 ring heteroatoms provided in thearomatic ring system, wherein each heteroatom is independently selectedfrom nitrogen, oxygen, and sulfur (“5-6 membered heteroaryl”). In someembodiments, the 5-6 membered heteroaryl has 1-3 ring heteroatomsselected from nitrogen, oxygen, and sulfur. In some embodiments, the 5-6membered heteroaryl has 1-2 ring heteroatoms selected from nitrogen,oxygen, and sulfur. In some embodiments, the 5-6 membered heteroaryl has1 ring heteroatom selected from nitrogen, oxygen, and sulfur. Unlessotherwise specified, each instance of a heteroaryl group isindependently unsubstituted (an “unsubstituted heteroaryl”) orsubstituted (a “substituted heteroaryl”) with one or more substituents.In certain embodiments, the heteroaryl group is an unsubstituted 5-14membered heteroaryl. In certain embodiments, the heteroaryl group is asubstituted 5-14 membered heteroaryl.

Exemplary 5-membered heteroaryl groups containing 1 heteroatom include,without limitation, pyrrolyl, furanyl, and thiophenyl. Exemplary5-membered heteroaryl groups containing 2 heteroatoms include, withoutlimitation, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, thiazolyl, andisothiazolyl. Exemplary 5-membered heteroaryl groups containing 3heteroatoms include, without limitation, triazolyl, oxadiazolyl, andthiadiazolyl. Exemplary 5-membered heteroaryl groups containing 4heteroatoms include, without limitation, tetrazolyl. Exemplary6-membered heteroaryl groups containing 1 heteroatom include, withoutlimitation, pyridinyl. Exemplary 6-membered heteroaryl groups containing2 heteroatoms include, without limitation, pyridazinyl, pyrimidinyl, andpyrazinyl. Exemplary 6-membered heteroaryl groups containing 3 or 4heteroatoms include, without limitation, triazinyl and tetrazinyl,respectively. Exemplary 7-membered heteroaryl groups containing 1heteroatom include, without limitation, azepinyl, oxepinyl, andthiepinyl. Exemplary 5,6-bicyclic heteroaryl groups include, withoutlimitation, indolyl, isoindolyl, indazolyl, benzotriazolyl,benzothiophenyl, isobenzothiophenyl, benzofuranyl, benzoisofuranyl,benzimidazolyl, benzoxazolyl, benzisoxazolyl, benzoxadiazolyl,benzthiazolyl, benzisothiazolyl, benzthiadiazolyl, indolizinyl, andpurinyl. Exemplary 6,6-bicyclic heteroaryl groups include, withoutlimitation, naphthyridinyl, pteridinyl, quinolinyl, isoquinolinyl,cinnolinyl, quinoxalinyl, phthalazinyl, and quinazolinyl. Exemplarytricyclic heteroaryl groups include, without limitation,phenanthridinyl, dibenzofuranyl, carbazolyl, acridinyl, phenothiazinyl,phenoxazinyl, and phenazinyl.

Affixing the suffix “-ene” to a group indicates the group is a divalentmoiety, e.g., alkylene is the divalent moiety of alkyl, alkenylene isthe divalent moiety of alkenyl, alkynylene is the divalent moiety ofalkynyl, heteroalkylene is the divalent moiety of heteroalkyl,heteroalkenylene is the divalent moiety of heteroalkenyl,heteroalkynylene is the divalent moiety of heteroalkynyl, carbocyclyleneis the divalent moiety of carbocyclyl, heterocyclylene is the divalentmoiety of heterocyclyl, arylene is the divalent moiety of aryl, andheteroarylene is the divalent moiety of heteroaryl.

A group is optionally substituted unless expressly provided otherwise.The term “optionally substituted” refers to being substituted orunsubstituted. In certain embodiments, alkyl, alkenyl, alkynyl,heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl,aryl, and heteroaryl groups are optionally substituted. “Optionallysubstituted” refers to a group which may be substituted or unsubstituted(e.g., “substituted” or “unsubstituted” alkyl, “substituted” or“unsubstituted” alkenyl, “substituted” or “unsubstituted” alkynyl,“substituted” or “unsubstituted” heteroalkyl, “substituted” or“unsubstituted” heteroalkenyl, “substituted” or “unsubstituted”heteroalkynyl, “substituted” or “unsubstituted” carbocyclyl,“substituted” or “unsubstituted” heterocyclyl, “substituted” or“unsubstituted” aryl or “substituted” or “unsubstituted” heteroarylgroup). In general, the term “substituted” means that at least onehydrogen present on a group is replaced with a permissible substituent,e.g., a substituent which upon substitution results in a stablecompound, e.g., a compound which does not spontaneously undergotransformation such as by rearrangement, cyclization, elimination, orother reaction. Unless otherwise indicated, a “substituted” group has asubstituent at one or more substitutable positions of the group, andwhen more than one position in any given structure is substituted, thesubstituent is either the same or different at each position. The term“substituted” is contemplated to include substitution with allpermissible substituents of organic compounds, and includes any of thesubstituents described herein that results in the formation of a stablecompound. The present invention contemplates any and all suchcombinations in order to arrive at a stable compound. For purposes ofthis invention, heteroatoms such as nitrogen may have hydrogensubstituents and/or any suitable substituent as described herein whichsatisfy the valencies of the heteroatoms and results in the formation ofa stable moiety. The invention is not intended to be limited in anymanner by the exemplary substituents described herein.

Exemplary carbon atom substituents include, but are not limited to,halogen, —CN, —NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(aa), —ON(R^(bb))₂,—N(R^(bb))₂, —N(R^(bb))₃ ⁺X⁻, —N(OR^(cc))R^(bb), —SH, —SR^(aa),—SSR^(cc), —C(═O)R^(aa), —CO₂H, —CHO, —C(OR^(cc))₃, —CO₂R^(aa),—OC(═O)R^(aa), —OCO₂R^(aa), —C(═O)N(R^(bb))₂, —OC(═O)N(R^(bb))₂,—NR^(bb)C(═O)R^(aa), —NR^(bb)CO₂R^(aa), —NR^(bb)C(═O)N(R^(bb))₂,—C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa), —OC(═NR^(bb))R^(aa),—OC(═NR^(bb))OR^(aa), —C(═NR^(bb))N(R^(bb))₂,—OC(═NR^(bb))N(R^(bb))₂—NR^(bb)C(═NR^(bb))N(R^(bb))₂,—C(═O)NR^(bb)SO₂R^(aa), —NR^(bb)SO₂R^(aa), —SO₂N(R^(bb))₂, —SO₂R^(aa),—SO₂OR^(aa), —OSO₂R^(aa), —S(═O)R^(aa), —OS(═O)R^(aa), —Si(R^(aa))₃,—OSi(R^(aa))₃—C(═S)N(R^(bb))₂, —C(═O)SR^(aa), —C(═S)SR^(aa),—SC(═S)SR^(aa), —SC(═O)SR^(aa), —OC(═O)SR^(aa), —SC(═O)OR^(aa),—SC(═O)R^(aa), —P(═O)(R^(aa))₂, —P(═O)(OR^(cc))₂, —OP(═O)(R^(aa))₂,—OP(═O)(OR^(cc))₂, —P(═O)(N(R^(bb))₂)₂, —OP(═O)(N(R^(bb))₂)₂,—NR^(bb)P(═O)(R^(aa))₂, —NR^(bb)P(═O)(OR^(cc))₂,—NR^(bb)P(═O)(N(R^(bb))₂)₂, —P(R^(cc))₂, —P(OR^(cc))₂, —P(R^(cc))₃ ⁺X⁻,—P(OR^(cc))₃ ⁺X⁻, —P(R^(cc))₄, —P(OR^(cc))₄, —OP(R^(cc))₂, —OP(R^(cc))₃⁺X⁻, —OP(OR^(cc))₂, —OP(OR^(cc))₃ ⁺X⁻, —OP(R^(cc))₄, —OP(OR^(cc))₄,—B(R^(aa))₂, —B(OR^(cc))₂, —BR^(aa)(OR^(cc)), C₁₋₁₀ alkyl, C₁₋₁₀perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroC₁₋₁₀ alkyl,heteroC₂₋₁₀ alkenyl, heteroC₂₋₁₀ alkynyl, C₃₋₁₀ carbocyclyl, 3-14membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, whereineach alkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups; wherein X⁻ is acounterion;

or two geminal hydrogens on a carbon atom are replaced with the group═O, ═S, ═NN(R^(bb))₂, ═NNR^(bb)C(═O)R^(aa), ═NNR^(bb)C(═O)OR^(aa),═NNR^(bb)S(═O)₂R^(aa), ═NR^(bb), or ═NOR^(cc);

each instance of R^(aa) is, independently, selected from C₁₋₁₀ alkyl,C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroC₁₋₁₀ alkyl,heteroC₂₋₁₀ alkenyl, heteroC₂₋₁₀ alkynyl, C₃₋₁₀ carbocyclyl, 3-14membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or twoR^(aa) groups are joined to form a 3-14 membered heterocyclyl or 5-14membered heteroaryl ring, wherein each alkyl, alkenyl, alkynyl,heteroalkyl, heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl,aryl, and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or5 R^(dd) groups;

-   -   each instance of R^(bb) is, independently, selected from        hydrogen, —OH, —OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa),        —C(═O)N(R^(cc))₂, —CO₂R^(aa), —SO₂R^(aa), —C(═NR^(cc))OR^(aa),        —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc), —SO₂OR^(cc),        —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc),        —P(═O)(R^(aa))₂, —P(═O)(OR^(cc))₂, —P(═O)(N(R^(cc))₂)₂, C₁₋₁₀        alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,        heteroC₁₋₁₀ alkyl, heteroC₂₋₁₀ alkenyl, heteroC₂₋₁₀alkynyl,        C₃₋₁₀ carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and        5-14 membered heteroaryl, or two R^(bb) groups are joined to        form a 3-14 membered heterocyclyl or 5-14 membered heteroaryl        ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl,        heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl,        and heteroaryl is independently substituted with 0, 1, 2, 3, 4,        or 5 R^(dd) groups; wherein X⁻ is a counterion;

each instance of R^(cc) is, independently, selected from hydrogen,C₁₋₁₀alkyl, C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,heteroC₁₋₁₀ alkyl, heteroC₂₋₁₀ alkenyl, heteroC₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl, or two R^(cc) groups are joined to form a 3-14 memberedheterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl,alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups;

each instance of R^(dd) is, independently, selected from halogen, —CN,—NO₂, —N₃, —SO₂H, —SO₃H, —OH, —OR^(ee), —ON(R^(ff))₂, —N(R^(ff))₂,—N(R^(ff))₃ ⁺X⁻, —N(OR^(ee))R^(ff), —SH, —SR^(ee), —SSR^(ee),—C(═O)R^(ee), —CO₂H, —CO₂R^(ee), —OC(═O)R^(ee), —OCO₂R^(ee),—C(═O)N(R^(ff))₂, —OC(═O)N(R^(ff))₂, —NR^(ff)C(═O)R^(ee),—NR^(ff)CO₂R^(ee), —NR^(ff)C(═O)N(R^(ff))₂, —C(═NR^(ff))OR^(ee),—OC(═NR^(ff))R^(ee), —OC(═NR^(ff))OR^(ee), —C(═NR^(ff))N(R^(ff))₂,—OC(═NR^(ff))N(R^(ff))₂, —NR^(ff)C(═NR^(ff))N(R^(ff))₂,—NR^(ff)SO₂R^(ee), —SO₂N(R^(ff))₂, —SO₂R^(ee), —SO₂OR^(ee), —OSO₂R^(ee),—S(═O)R^(ee), —Si(R^(ee))₃, —OSi(R^(ee))₃, —C(═S)N(R^(ff))₂,—C(═O)SR^(ee), —C(═S)SR^(ee), —SC(═S)SR^(ee), —P(═O)(OR^(ee))₂,—P(═O)(R^(ee))₂, —OP(═O)(R^(ee))₂, —OP(═O)(OR^(ee))₂, C₁₋₆ alkyl, C₁₋₆perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, heteroC₁₋₆ alkyl, heteroC₂₋₆alkenyl, heteroC₂₋₆ alkynyl, C₃₋₁₀ carbocyclyl, 3-10 memberedheterocyclyl, C₆₋₁₀ aryl, 5-10 membered heteroaryl, wherein each alkyl,alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(gg) groups, or two geminalR^(dd) substituents can be joined to form ═O or ═S; wherein X⁻ is acounterion;

each instance of R^(ee) is, independently, selected from C₁₋₆ alkyl,C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, heteroC₁₋₆ alkyl,heteroC₂₋₆ alkenyl, heteroC₂₋₆ alkynyl, C₃₋₁₀ carbocyclyl, C₆₋₁₀ aryl,3-10 membered heterocyclyl, and 3-10 membered heteroaryl, wherein eachalkyl, alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(gg) groups;

each instance of R^(ff) is, independently, selected from hydrogen, C₁₋₆alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, heteroC₁₋₆ alkyl,heteroC₂₋₆ alkenyl, heteroC₂₋₆ alkynyl, C₃₋₁₀ carbocyclyl, 3-10 memberedheterocyclyl, C₆₋₁₀ aryl and 5-10 membered heteroaryl, or two R^(f)groups are joined to form a 3-10 membered heterocyclyl or 5-10 memberedheteroaryl ring, wherein each alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aryl, andheteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5 R^(gg)groups; and

each instance of R^(gg) is, independently, halogen, —CN, —NO₂, —N₃,—SO₂H, —SO₃H, —OH, —OC₁₋₆ alkyl, —ON(C₁₋₆ alkyl)₂, —N(C₁₋₆ alkyl)₂,—N(C₁₋₆ alkyl)₃ ⁺X⁻, —NH(C₁₋₆ alkyl)₂ ⁺X⁻, —NH₂(C₁₋₆ alkyl)⁺X⁻, —NH₃⁺X⁻, —N(OC₁₋₆ alkyl)(C₁₋₆ alkyl), —N(OH)(C₁₋₆ alkyl), —NH(OH), —SH,—SC₁₋₆ alkyl, —SS(C₁₋₆ alkyl), —C(═O)(C₁₋₆ alkyl), —CO₂H, —CO₂(C₁₋₆alkyl), —OC(═O)(C₁₋₆ alkyl), —OCO₂(C₁₋₆ alkyl), —C(═O)NH₂, —C(═O)N(C₁₋₆alkyl)₂, —OC(═O)NH(C₁₋₆ alkyl), —NHC(═O)(C₁₋₆ alkyl), —N(C₁₋₆alkyl)C(═O)(C₁₋₆ alkyl), —NHCO₂(C₁₋₆ alkyl), —NHC(═O)N(C₁₋₆ alkyl)₂,—NHC(═O)NH(C₁₋₆ alkyl), —NHC(═O)NH₂, —C(═NH)O(C₁₋₆ alkyl), —OC(═NH)(C₁₋₆alkyl), —OC(═NH)OC₁₋₆ alkyl, —C(═NH)N(C₁₋₆ alkyl)₂, —C(═NH)NH(C₁₋₆alkyl), —C(═NH)NH₂, —OC(═NH)N(C₁₋₆ alkyl)₂, —OC(═NH)NH(C₁₋₆ alkyl),—OC(═NH)NH₂, —NHC(═NH)N(C₁₋₆ alkyl)₂, —NHC(═NH)NH₂, —NHSO₂(C₁₋₆ alkyl),—SO₂N(C₁₋₆ alkyl)₂, —SO₂NH(C₁₋₆ alkyl), —SO₂NH₂, —SO₂(C₁₋₆ alkyl),—SO₂O(C₁₋₆ alkyl), —OSO₂(C₁₋₆ alkyl), —SO(C₁₋₆ alkyl), —Si(C₁₋₆ alkyl)₃,—OSi(C₁₋₆ alkyl)₃—C(═S)N(C₁₋₆ alkyl)₂, C(═S)NH(C₁₋₆ alkyl), C(═S)NH₂,—C(═O)S(C₁₋₆ alkyl), —C(═S)SC₁₋₆ alkyl, —SC(═S)SC₁₋₆ alkyl, —P(═O)(OC₁₋₆alkyl)₂, —P(═O)(C₁₋₆ alkyl)₂, —OP(═O)(C₁₋₆ alkyl)₂, —OP(═O)(OC₁₋₆alkyl)₂, C₁₋₆ alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,heteroC₁₋₆ alkyl, heteroC₂₋₆ alkenyl, heteroC₂₋₆ alkynyl, C₃₋₁₀carbocyclyl, C₆₋₁₀ aryl, 3-10 membered heterocyclyl, 5-10 memberedheteroaryl; or two geminal R^(gg) substituents can be joined to form ═Oor ═S; wherein X⁻ is a counterion.

In certain embodiments, substituents include: halogen, —CN, —NO₂, —N₃,—SO₂H, —SO₃H, —OH, —OC₁₋₆ alkyl, —ON(C₁₋₆ alkyl)₂, —N(C₁₋₆ alkyl)₂,—N(C₁₋₆ alkyl)₃ ⁺X⁻, —NH(C₁₋₆ alkyl)₂ ⁺X⁻, —NH₂(C₁₋₆ alkyl)⁺X⁻, —NH₃⁺X⁻, —N(OC₁₋₆ alkyl)(C₁₋₆ alkyl), —N(OH)(C₁₋₆ alkyl), —NH(OH), —SH,—SC₁₋₆ alkyl, —SS(C₁₋₆ alkyl), —C(═O)(C₁₋₆ alkyl), —CO₂H, —CO₂(C₁₋₆alkyl), —OC(═O)(C₁₋₆ alkyl), —OCO₂(C₁₋₆ alkyl), —C(═O)NH₂, —C(═O)N(C₁₋₆alkyl)₂, —OC(═O)NH(C₁₋₆ alkyl), —NHC(═O)(C₁₋₆ alkyl), —N(C₁₋₆alkyl)C(═O)(C₁₋₆ alkyl), —NHCO₂(C₁₋₆ alkyl), —NHC(═O)N(C₁₋₆ alkyl)₂,—NHC(═O)NH(C₁₋₆ alkyl), —NHC(═O)NH₂, —C(═NH)O(C₁₋₆ alkyl), —OC(═NH)(C₁₋₆alkyl), —OC(═NH)OC₁₋₆ alkyl, —C(═NH)N(C₁₋₆ alkyl)₂, —C(═NH)NH(C₁₋₆alkyl), —C(═NH)NH₂, —OC(═NH)N(C₁₋₆ alkyl)₂, —OC(═NH)NH(C₁₋₆ alkyl),—OC(═NH)NH₂, —NHC(═NH)N(C₁₋₆ alkyl)₂, —NHC(═NH)NH₂, —NHSO₂(C₁₋₆ alkyl),—SO₂N(C₁₋₆ alkyl)₂, —SO₂NH(C₁₋₆ alkyl), —SO₂NH₂, —SO₂(C₁₋₆ alkyl),—SO₂O(C₁₋₆ alkyl), —OSO₂(C₁₋₆ alkyl), —SO(C₁₋₆ alkyl), —Si(C₁₋₆ alkyl)₃,—OSi(C₁₋₆ alkyl)₃—C(═S)N(C₁₋₆ alkyl)₂, C(═S)NH(C₁₋₆ alkyl), C(═S)NH₂,—C(═O)S(C₁₋₆ alkyl), —C(═S)SC₁₋₆ alkyl, —SC(═S)SC₁₋₆ alkyl, —P(═O)(OC₁₋₆alkyl)₂, —P(═O)(C₁₋₆ alkyl)₂, —OP(═O)(C₁₋₆ alkyl)₂, —OP(═O)(OC₁₋₆alkyl)₂, C₁₋₆ alkyl, C₁₋₆ perhaloalkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl,heteroC₁₋₆ alkyl, heteroC₂₋₆ alkenyl, heteroC₂₋₆ alkynyl, C₃₋₁₀carbocyclyl, C₆₋₁₀ aryl, 3-10 membered heterocyclyl, 5-10 memberedheteroaryl; or two geminal hydrogens can be joined to form ═O or ═S;wherein X⁺ is a counterion.

The term “halo” or “halogen” refers to fluorine (fluoro, —F), chlorine(chloro, —Cl), bromine (bromo, —Br), or iodine (iodo, —I).

The term “hydroxyl” or “hydroxy” refers to the group —OH. The term“substituted hydroxyl” or “substituted hydroxyl,” by extension, refersto a hydroxyl group wherein the oxygen atom directly attached to theparent molecule is substituted with a group other than hydrogen, andincludes groups selected from —OR^(aa), —ON(R^(bb))₂, —OC(═O)SR^(aa),—OC(═O)R^(aa), —OCO₂R^(aa), —OC(═O)N(R^(bb))₂, —OC(═NR^(bb))R^(aa),—OC(═NR^(bb))OR^(aa), —OC(═NR^(bb))N(R^(bb))₂, —OS(═O)R^(aa),—OSO₂R^(aa), —OSi(R^(aa))₃, —OP(R^(cc))₂, —OP(R^(cc))₃ ⁺X⁻, —OP(OR)₂,—OP(OR^(cc))₃ ⁺X⁻, —OP(═O)(R^(aa))₂, —OP(═O)(OR^(cc))₂, and—OP(═O)(N(R^(bb))₂)₂, wherein X⁻, R^(aa), R^(bb), and R^(cc) are asdefined herein.

The term “amino” refers to the group —NH₂. The term “substituted amino,”by extension, refers to a monosubstituted amino, a disubstituted amino,or a trisubstituted amino.

In certain embodiments, the “substituted amino” is a monosubstitutedamino or a disubstituted amino group.

The term “monosubstituted amino” refers to an amino group wherein thenitrogen atom directly attached to the parent molecule is substitutedwith one hydrogen and one group other than hydrogen, and includes groupsselected from —NH(R^(bb)), —NHC(═O)R^(aa), —NHCO₂R^(aa),—NHC(═O)N(R^(bb))₂, —NHC(═NR^(bb))N(R^(bb))₂, —NHSO₂R^(aa),—NHP(═O)(OR^(cc))₂, and —NHP(═O)(N(R^(bb))₂)₂, wherein R^(aa), R^(bb)and R^(cc) are as defined herein, and wherein R^(bb) of the group—NH(R^(bb)) is not hydrogen.

The term “disubstituted amino” refers to an amino group wherein thenitrogen atom directly attached to the parent molecule is substitutedwith two groups other than hydrogen, and includes groups selected from—N(R^(bb))₂, —NR^(bb)C(═O)R, —NR^(bb)CO₂R^(aa), —NR^(bb)C(═O)N(R^(bb))₂,—NR^(bb)C(═NR^(bb))N(R^(bb))₂—NR^(bb)SO₂R^(aa), —NR^(bb)P(═O)(OR^(cc))₂,and —NR^(bb)P(═O)(N(R^(bb))₂)₂, wherein R^(a), R^(bb), and R^(cc) are asdefined herein, with the proviso that the nitrogen atom directlyattached to the parent molecule is not substituted with hydrogen.

The term “trisubstituted amino” refers to an amino group wherein thenitrogen atom directly attached to the parent molecule is substitutedwith three groups, and includes groups selected from —N(R^(bb))₃ and—N(R^(bb))₃ ⁺X⁻, wherein R^(bb) and X⁻ are as defined herein.

The term “sulfonyl” refers to a group selected from —SO₂N(R^(bb))₂,—SO₂R^(aa), and —SO₂OR^(aa), wherein R^(aa) and R^(bb) are as definedherein.

The term “sulfinyl” refers to the group —S(═O)R^(aa), wherein R^(aa) isas defined herein.

The term “acyl” refers to a group having the general formula—C^((═O))R^(X1), —C(═O)OR^(X1), —C(═O)—O—C(═O)R^(X1), —C(═O)SR,—C(═O)N(R^(X1))₂, —C(═S)R^(X1), —C(═S)N(R^(X1))₂, —C(═S)O(R^(X1)),—C(═S)S(R^(X1)), —C(═NR^(X1))R^(X1), —C(═NR^(X1))OR^(X1),—C(═NR^(X1))SR^(X1), and —C(═NR^(X1))N(R^(X1))₂, wherein R^(X1) ishydrogen; halogen; substituted or unsubstituted hydroxyl; substituted orunsubstituted thiol; substituted or unsubstituted amino; substituted orunsubstituted acyl, cyclic or acyclic, substituted or unsubstituted,branched or unbranched aliphatic; cyclic or acyclic, substituted orunsubstituted, branched or unbranched heteroaliphatic; cyclic oracyclic, substituted or unsubstituted, branched or unbranched alkyl;cyclic or acyclic, substituted or unsubstituted, branched or unbranchedalkenyl; substituted or unsubstituted alkynyl; substituted orunsubstituted aryl, substituted or unsubstituted heteroaryl,aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy, aryloxy,heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy, alkylthioxy,heteroalkylthioxy, arylthioxy, heteroarylthioxy, mono- ordi-aliphaticamino, mono- or di-heteroaliphaticamino, mono- ordi-alkylamino, mono- or di-heteroalkylamino, mono- or di-arylamino, ormono- or di-heteroarylamino; or two R^(X1) groups taken together form a5- to 6-membered heterocyclic ring. Exemplary acyl groups includealdehydes (—CHO), carboxylic acids (—CO₂H), ketones, acyl halides,esters, amides, imines, carbonates, carbamates, and ureas. Acylsubstituents include, but are not limited to, any of the substituentsdescribed herein, that result in the formation of a stable moiety (e.g.,aliphatic, alkyl, alkenyl, alkynyl, heteroaliphatic, heterocyclic, aryl,heteroaryl, acyl, oxo, imino, thiooxo, cyano, isocyano, amino, azido,nitro, hydroxyl, thiol, halo, aliphaticamino, heteroaliphaticamino,alkylamino, heteroalkylamino, arylamino, heteroarylamino, alkylaryl,arylalkyl, aliphaticoxy, heteroaliphaticoxy, alkyloxy, heteroalkyloxy,aryloxy, heteroaryloxy, aliphaticthioxy, heteroaliphaticthioxy,alkylthioxy, heteroalkylthioxy, arylthioxy, heteroarylthioxy, acyloxy,and the like, each of which may or may not be further substituted).

The term “carbonyl” refers a group wherein the carbon directly attachedto the parent molecule is sp² hybridized, and is substituted with anoxygen, nitrogen or sulfur atom, e.g., a group selected from ketones(e.g., —C(═O)R^(aa)), carboxylic acids (e.g., —CO₂H), aldehydes (—CHO),esters (e.g., —CO₂R^(aa), —C(═O)SR^(aa), —C(═S)SR^(aa)), amides (e.g.,—C(═O)N(R^(bb))₂, —C(═O)NR^(bb)SO₂R^(aa), —C(═S)N(R^(bb))₂), and imines(e.g., —C(═NR^(bb))R^(aa), C(═NR^(bb))OR^(aa)), —C(═NR^(bb))N(R^(bb))₂),wherein R^(aa) and R^(bb) are as defined herein.

The term “silyl” refers to the group —Si(R^(aa))₃, wherein R^(aa) is asdefined herein.

The term “oxo” refers to the group ═O, and the term “thiooxo” refers tothe group ═S.

Nitrogen atoms can be substituted or unsubstituted as valency permits,and include primary, secondary, tertiary, and quaternary nitrogen atoms.Exemplary nitrogen atom substituents include, but are not limited to,hydrogen, —OH, —OR^(aa), —N(R^(cc))₂, —CN, —C(═O)R^(aa),—C(═O)N(R^(cc))₂, —CO₂R^(aa), —SO₂R^(aa), —C(═NR^(bb))R^(aa),—C(═NR^(cc))OR^(aa), —C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc),—SO₂OR^(cc), —SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc),—P(═O)(OR^(cc))₂, —P(═O)(R^(aa))₂, —P(═O)(N(R^(cc))₂)₂, C₁₋₁₀ alkyl,C₁₋₁₀ perhaloalkyl, C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl, heteroC₁₋₁₀alkyl,heteroC₂₋₁₀alkenyl, heteroC₂₋₁₀alkynyl, C₃₋₁₀ carbocyclyl, 3-14 memberedheterocyclyl, C₆₋₁₄ aryl, and 5-14 membered heteroaryl, or two R^(cc)groups attached to an N atom are joined to form a 3-14 memberedheterocyclyl or 5-14 membered heteroaryl ring, wherein each alkyl,alkenyl, alkynyl, heteroalkyl, heteroalkenyl, heteroalkynyl,carbocyclyl, heterocyclyl, aryl, and heteroaryl is independentlysubstituted with 0, 1, 2, 3, 4, or 5 R^(dd) groups, and wherein R^(aa),R^(bb), R^(cc) and R^(dd) are as defined above.

In certain embodiments, the substituent present on the nitrogen atom isan nitrogen protecting group (also referred to herein as an “aminoprotecting group”). Nitrogen protecting groups include, but are notlimited to, —OH, —OR^(aa), —N(R^(cc))₂, —C(═O)R^(aa), —C(═O)N(R^(cc))₂,—CO₂R^(aa), —SO₂R^(aa), —C(═NR^(cc))R^(aa), —C(═NR^(cc))OR^(aa),—C(═NR^(cc))N(R^(cc))₂, —SO₂N(R^(cc))₂, —SO₂R^(cc), —SO₂OR^(cc),—SOR^(aa), —C(═S)N(R^(cc))₂, —C(═O)SR^(cc), —C(═S)SR^(cc), C₁₋₁₀ alkyl(e.g., aralkyl, heteroaralkyl), C₂₋₁₀ alkenyl, C₂₋₁₀ alkynyl,heteroC₁₋₁₀ alkyl, heteroC₂₋₁₀ alkenyl, heteroC₂₋₁₀ alkynyl, C₃₋₁₀carbocyclyl, 3-14 membered heterocyclyl, C₆₋₁₄ aryl, and 5-14 memberedheteroaryl groups, wherein each alkyl, alkenyl, alkynyl, heteroalkyl,heteroalkenyl, heteroalkynyl, carbocyclyl, heterocyclyl, aralkyl, aryl,and heteroaryl is independently substituted with 0, 1, 2, 3, 4, or 5R^(dd) groups, and wherein R^(aa), R^(bb), R^(cc) and Rad are as definedherein. Nitrogen protecting groups are well known in the art and includethose described in detail in Protecting Groups in Organic Synthesis, T.W. Greene and P. G. M. Wuts, 3^(rd) edition, John Wiley & Sons, 1999,incorporated herein by reference.

For example, nitrogen protecting groups such as amide groups (e.g.,—C(═O)R^(aa)) include, but are not limited to, formamide, acetamide,chloroacetamide, trichloroacetamide, trifluoroacetamide,phenylacetamide, 3-phenylpropanamide, picolinamide,3-pyridylcarboxamide, N-benzoylphenylalanyl derivative, benzamide,p-phenylbenzamide, o-nitophenylacetamide, o-nitrophenoxyacetamide,acetoacetamide, (N′-dithiobenzyloxyacylamino)acetamide,3-(p-hydroxyphenyl)propanamide, 3-(o-nitrophenyl)propanamide,2-methyl-2-(o-nitrophenoxy)propanamide,2-methyl-2-(o-phenylazophenoxy)propanamide, 4-chlorobutanamide,3-methyl-3-nitrobutanamide, o-nitrocinnamide, N-acetylmethioninederivative, o-nitrobenzamide and o-(benzoyloxymethyl)benzamide.

Nitrogen protecting groups such as carbamate groups (e.g.,—C(═O)OR^(aa)) include, but are not limited to, methyl carbamate, ethylcarbamate, 9-fluorenylmethyl carbamate (Fmoc),9-(2-sulfo)fluorenylmethyl carbamate, 9-(2,7-dibromo)fluoroenylmethylcarbamate,2,7-di-t-butyl-[9-(10,10-dioxo-10,10,10,10-tetrahydrothioxanthyl)]methylcarbamate (DBD-Tmoc), 4-methoxyphenacyl carbamate (Phenoc),2,2,2-trichloroethyl carbamate (Troc), 2-trimethylsilylethyl carbamate(Teoc), 2-phenylethyl carbamate (hZ), 1-(1-adamantyl)-1-methylethylcarbamate (Adpoc), 1,1-dimethyl-2-haloethyl carbamate,1,1-dimethyl-2,2-dibromoethyl carbamate (DB-t-BOC),1,1-dimethyl-2,2,2-trichloroethyl carbamate (TCBOC),1-methyl-1-(4-biphenylyl)ethyl carbamate (Bpoc),1-(3,5-di-t-butylphenyl)-1-methylethyl carbamate (t-Bumeoc), 2-(2′- and4′-pyridyl)ethyl carbamate (Pyoc), 2-(N,N-dicyclohexylcarboxamido)ethylcarbamate, t-butyl carbamate (BOC or Boc), 1-adamantyl carbamate (Adoc),vinyl carbamate (Voc), allyl carbamate (Alloc), 1-isopropylallylcarbamate (Ipaoc), cinnamyl carbamate (Coc), 4-nitrocinnamyl carbamate(Noc), 8-quinolyl carbamate, N-hydroxypiperidinyl carbamate, alkyldithiocarbamate, benzyl carbamate (Cbz), p-methoxybenzyl carbamate (Moz),p-nitrobenzyl carbamate, p-bromobenzyl carbamate, p-chlorobenzylcarbamate, 2,4-dichlorobenzyl carbamate, 4-methylsulfinylbenzylcarbamate (Msz), 9-anthrylmethyl carbamate, diphenylmethyl carbamate,2-methylthioethyl carbamate, 2-methylsulfonylethyl carbamate,2-(p-toluenesulfonyl)ethyl carbamate, [2-(1,3-dithianyl)]methylcarbamate (Dmoc), 4-methylthiophenyl carbamate (Mtpc),2,4-dimethylthiophenyl carbamate (Bmpc), 2-phosphonioethyl carbamate(Peoc), 2-triphenylphosphonioisopropyl carbamate (Ppoc),1,1-dimethyl-2-cyanoethyl carbamate, m-chloro-p-acyloxybenzyl carbamate,p-(dihydroxyboryl)benzyl carbamate, 5-benzisoxazolylmethyl carbamate,2-(trifluoromethyl)-6-chromonylmethyl carbamate (Tcroc), m-nitrophenylcarbamate, 3,5-dimethoxybenzyl carbamate, o-nitrobenzyl carbamate,3,4-dimethoxy-6-nitrobenzyl carbamate, phenyl(o-nitrophenyl)methylcarbamate, t-amyl carbamate, S-benzyl thiocarbamate, p-cyanobenzylcarbamate, cyclobutyl carbamate, cyclohexyl carbamate, cyclopentylcarbamate, cyclopropylmethyl carbamate, p-decyloxybenzyl carbamate,2,2-dimethoxyacylvinyl carbamate, o-(N,N-dimethylcarboxamido)benzylcarbamate, 1,1-dimethyl-3-(N,N-dimethylcarboxamido)propyl carbamate,1,1-dimethylpropynyl carbamate, di(2-pyridyl)methyl carbamate,2-furanylmethyl carbamate, 2-iodoethyl carbamate, isoborynl carbamate,isobutyl carbamate, isonicotinyl carbamate,p-(p′-methoxyphenylazo)benzyl carbamate, 1-methylcyclobutyl carbamate,1-methylcyclohexyl carbamate, 1-methyl-1-cyclopropylmethyl carbamate,1-methyl-1-(3,5-dimethoxyphenyl)ethyl carbamate,1-methyl-1-(p-phenylazophenyl)ethyl carbamate, 1-methyl-1-phenylethylcarbamate, 1-methyl-1-(4-pyridyl)ethyl carbamate, phenyl carbamate,p-(phenylazo)benzyl carbamate, 2,4,6-tri-t-butylphenyl carbamate,4-(trimethylammonium)benzyl carbamate, and 2,4,6-trimethylbenzylcarbamate.

Nitrogen protecting groups such as sulfonamide groups (e.g.,—S(═O)₂R^(aa)) include, but are not limited to, p-toluenesulfonamide(Ts), benzenesulfonamide, 2,3,6-trimethyl-4-methoxybenzenesulfonamide(Mtr), 2,4,6-trimethoxybenzenesulfonamide (Mtb),2,6-dimethyl-4-methoxybenzenesulfonamide (Pme),2,3,5,6-tetramethyl-4-methoxybenzenesulfonamide (Mte),4-methoxybenzenesulfonamide (Mbs), 2,4,6-trimethylbenzenesulfonamide(Mts), 2,6-dimethoxy-4-methylbenzenesulfonamide (iMds),2,2,5,7,8-pentamethylchroman-6-sulfonamide (Pme), methanesulfonamide(Ms), (3-trimethylsilylethanesulfonamide (SES), 9-anthracenesulfonamide,4-(4′,8′-dimethoxynaphthylmethyl)benzenesulfonamide (DNMBS),benzylsulfonamide, trifluoromethylsulfonamide, and phenacylsulfonamide.

Other nitrogen protecting groups include, but are not limited to,phenothiazinyl-(10)-acyl derivative, N′-p-toluenesulfonylaminoacylderivative, N′-phenylaminothioacyl derivative, N-benzoylphenylalanylderivative, N-acetylmethionine derivative,4,5-diphenyl-3-oxazolin-2-one, N-phthalimide, N-dithiasuccinimide (Dts),N-2,3-diphenylmaleimide, N-2,5-dimethylpyrrole,N-1,1,4,4-tetramethyldisilylazacyclopentane adduct (STABASE),5-substituted 1,3-dimethyl-1,3,5-triazacyclohexan-2-one, 5-substituted1,3-dibenzyl-1,3,5-triazacyclohexan-2-one, 1-substituted3,5-dinitro-4-pyridone, N-methylamine, N-allylamine,N-[2-(trimethylsilyl)ethoxy]methylamine (SEM), N-3-acetoxypropylamine,N-(1-isopropyl-4-nitro-2-oxo-3-pyroolin-3-yl)amine, quaternary ammoniumsalts, N-benzylamine, N-di(4-methoxyphenyl)methylamine,N—S-dibenzosuberylamine, N-triphenylmethylamine (Tr),N-[(4-methoxyphenyl)diphenylmethyl]amine (MMTr),N-9-phenylfluorenylamine (PhF),N-2,7-dichloro-9-fluorenylmethyleneamine, N-ferrocenylmethylamino (Fcm),N-2-picolylamino N′-oxide, N-1,1-dimethylthiomethyleneamine,N-benzylideneamine, N-p-methoxybenzylideneamine,N-diphenylmethyleneamine, N-[(2-pyridyl)mesityl]methyleneamine,N—(N′,N′-dimethylaminomethylene)amine, N,N′-isopropylidenediamine,N-p-nitrobenzylideneamine, N-salicylideneamine,N—S-chlorosalicylideneamine,N-(5-chloro-2-hydroxyphenyl)phenylmethyleneamine,N-cyclohexylideneamine, N-(5,5-dimethyl-3-oxo-1-cyclohexenyl)amine,N-borane derivative, N-diphenylborinic acid derivative,N-[phenyl(pentaacylchromium- or tungsten)acyl]amine, N-copper chelate,N-zinc chelate, N-nitroamine, N-nitrosoamine, amine N-oxide,diphenylphosphinamide (Dpp), dimethylthiophosphinamide (Mpt),diphenylthiophosphinamide (Ppt), dialkyl phosphoramidates, dibenzylphosphoramidate, diphenyl phosphoramidate, benzenesulfenamide,o-nitrobenzenesulfenamide (Nps), 2,4-dinitrobenzenesulfenamide,pentachlorobenzenesulfenamide, 2-nitro-4-methoxybenzenesulfenamide,triphenylmethylsulfenamide, and 3-nitropyridinesulfenamide (Npys). Incertain embodiments, a nitrogen protecting group is benzyl (Bn),tert-butyloxycarbonyl (BOC), carbobenzyloxy (Cbz),9-flurenylmethyloxycarbonyl (Fmoc), trifluoroacetyl, triphenylmethyl,acetyl (Ac), benzoyl (Bz), p-methoxybenzyl (PMB), 3,4-dimethoxybenzyl(DMPM), p-methoxyphenyl (PMP), 2,2,2-trichloroethyloxycarbonyl (Troc),triphenylmethyl (Tr), tosyl (Ts), brosyl (Bs), nosyl (Ns), mesyl (Ms),triflyl (Tf), or dansyl (Ds).

In certain embodiments, the substituent present on an oxygen atom is anoxygen protecting group (also referred to herein as an “hydroxylprotecting group”). Oxygen protecting groups include, but are notlimited to, —R^(aa), —N(R^(bb))₂, —C(═O)SR^(aa), —C(═O)R^(aa),—CO₂R^(aa), —C(═O)N(R^(bb))₂, —C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa),—C(═NR^(bb))N(R^(bb))₂, —S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃,—P(R^(cc))₂, —P(R^(cc))₃ ⁺X⁻, —P(OR^(cc))₂, —P(OR^(cc))₃ ⁺X⁻,—P(═O)(R^(aa))₂, —P(═O)(OR^(cc))₂, and —P(═O)(N(R^(bb))₂)₂, wherein X⁻,R^(aa), R^(bb), and R^(cc) are as defined herein. Oxygen protectinggroups are well known in the art and include those described in detailin Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M.Wuts, 3^(rd) edition, John Wiley & Sons, 1999, incorporated herein byreference.

Exemplary oxygen protecting groups include, but are not limited to,methyl, methoxylmethyl (MOM), methylthiomethyl (MTM), t-butylthiomethyl,(phenyldimethylsilyl)methoxymethyl (SMOM), benzyloxymethyl (BOM),p-methoxybenzyloxymethyl (PMBM), (4-methoxyphenoxy)methyl (p-AOM),guaiacolmethyl (GUM), t-butoxymethyl, 4-pentenyloxymethyl (POM),siloxymethyl, 2-methoxyethoxymethyl (MEM), 2,2,2-trichloroethoxymethyl,bis(2-chloroethoxy)methyl, 2-(trimethylsilyl)ethoxymethyl (SEMOR),tetrahydropyranyl (THP), 3-bromotetrahydropyranyl,tetrahydrothiopyranyl, 1-methoxycyclohexyl, 4-methoxytetrahydropyranyl(MTHP), 4-methoxytetrahydrothiopyranyl, 4-methoxytetrahydrothiopyranylS,S-dioxide, 1-[(2-chloro-4-methyl)phenyl]-4-methoxypiperidin-4-yl(CTMP), 1,4-dioxan-2-yl, tetrahydrofuranyl, tetrahydrothiofuranyl,2,3,3a,4,5,6,7,7a-octahydro-7,8,8-trimethyl-4,7-methanobenzofuran-2-yl,1-ethoxyethyl, 1-(2-chloroethoxy)ethyl, 1-methyl-1-methoxyethyl,1-methyl-1-benzyloxyethyl, 1-methyl-1-benzyloxy-2-fluoroethyl,2,2,2-trichloroethyl, 2-trimethylsilylethyl, 2-(phenylselenyl)ethyl,t-butyl, allyl, p-chlorophenyl, p-methoxyphenyl, 2,4-dinitrophenyl,benzyl (Bn), p-methoxybenzyl, 3,4-dimethoxybenzyl, o-nitrobenzyl,p-nitrobenzyl, p-halobenzyl, 2,6-dichlorobenzyl, p-cyanobenzyl,p-phenylbenzyl, 2-picolyl, 4-picolyl, 3-methyl-2-picolyl N-oxido,diphenylmethyl, p,p′-dinitrobenzhydryl, 5-dibenzosuberyl,triphenylmethyl, α-naphthyldiphenylmethyl,p-methoxyphenyldiphenylmethyl, di(p-methoxyphenyl)phenylmethyl,tri(p-methoxyphenyl)methyl, 4-(4′-bromophenacyloxyphenyl)diphenylmethyl,4,4′,4″-tris(4,5-dichlorophthalimidophenyl)methyl,4,4′,4″-tris(levulinoyloxyphenyl)methyl,4,4′,4″-tris(benzoyloxyphenyl)methyl,3-(imidazol-1-yl)bis(4′,4″-dimethoxyphenyl)methyl,1,1-bis(4-methoxyphenyl)-1′-pyrenylmethyl, 9-anthryl,9-(9-phenyl)xanthenyl, 9-(9-phenyl-10-oxo)anthryl,1,3-benzodithiolan-2-yl, benzisothiazolyl S,S-dioxido, trimethylsilyl(TMS), triethylsilyl (TES), triisopropylsilyl (TIPS),dimethylisopropylsilyl (IPDMS), diethylisopropylsilyl (DEIPS),dimethylthexylsilyl, t-butyldimethylsilyl (TBDMS), t-butyldiphenylsilyl(TBDPS), tribenzylsilyl, tri-p-xylylsilyl, triphenylsilyl,diphenylmethylsilyl (DPMS), t-butylmethoxyphenylsilyl (TBMPS), formate,benzoylformate, acetate, chloroacetate, dichloroacetate,trichloroacetate, trifluoroacetate, methoxyacetate,triphenylmethoxyacetate, phenoxyacetate, p-chlorophenoxyacetate,3-phenylpropionate, 4-oxopentanoate (levulinate),4,4-(ethylenedithio)pentanoate (levulinoyldithioacetal), pivaloate,adamantoate, crotonate, 4-methoxycrotonate, benzoate, p-phenylbenzoate,2,4,6-trimethylbenzoate (mesitoate), methyl carbonate, 9-fluorenylmethylcarbonate (Fmoc), ethyl carbonate, 2,2,2-trichloroethyl carbonate(Troc), 2-(trimethylsilyl)ethyl carbonate (TMSEC), 2-(phenylsulfonyl)ethyl carbonate (Psec), 2-(triphenylphosphonio) ethyl carbonate (Peoc),isobutyl carbonate, vinyl carbonate, allyl carbonate, t-butyl carbonate(BOC or Boc), p-nitrophenyl carbonate, benzyl carbonate, p-methoxybenzylcarbonate, 3,4-dimethoxybenzyl carbonate, o-nitrobenzyl carbonate,p-nitrobenzyl carbonate, S-benzyl thiocarbonate, 4-ethoxy-1-napththylcarbonate, methyl dithiocarbonate, 2-iodobenzoate, 4-azidobutyrate,4-nitro-4-methylpentanoate, o-(dibromomethyl)benzoate,2-formylbenzenesulfonate, 2-(methylthiomethoxy)ethyl,4-(methylthiomethoxy)butyrate, 2-(methylthiomethoxymethyl)benzoate,2,6-dichloro-4-methylphenoxyacetate,2,6-dichloro-4-(1,1,3,3-tetramethylbutyl)phenoxyacetate,2,4-bis(1,1-dimethylpropyl)phenoxyacetate, chlorodiphenylacetate,isobutyrate, monosuccinoate, (E)-2-methyl-2-butenoate,o-(methoxyacyl)benzoate, α-naphthoate, nitrate, alkylN,N,N′,N′-tetramethylphosphorodiamidate, alkyl N-phenylcarbamate,borate, dimethylphosphinothioyl, alkyl 2,4-dinitrophenylsulfenate,sulfate, methanesulfonate (mesylate), benzylsulfonate, and tosylate(Ts). In certain embodiments, an oxygen protecting group is silyl. Incertain embodiments, an oxygen protecting group is t-butyldiphenylsilyl(TBDPS), t-butyldimethylsilyl (TBDMS), triisoproylsilyl (TIPS),triphenylsilyl (TPS), triethylsilyl (TES), trimethylsilyl (TMS),triisopropylsiloxymethyl (TOM), acetyl (Ac), benzoyl (Bz), allylcarbonate, 2,2,2-trichloroethyl carbonate (Troc), 2-trimethylsilylethylcarbonate, methoxymethyl (MOM), 1-ethoxyethyl (EE), 2-methyoxy-2-propyl(MOP), 2,2,2-trichloroethoxyethyl, 2-methoxyethoxymethyl (MEM),2-trimethylsilylethoxymethyl (SEM), methylthiomethyl (MTM),tetrahydropyranyl (THP), tetrahydrofuranyl (THF), p-methoxyphenyl (PMP),triphenylmethyl (Tr), methoxytrityl (MMT), dimethoxytrityl (DMT), allyl,p-methoxybenzyl (PMB), t-butyl, benzyl (Bn), allyl, or pivaloyl (Piv).

In certain embodiments, the substituent present on a sulfur atom is asulfur protecting group (also referred to as a “thiol protectinggroup”). Sulfur protecting groups include, but are not limited to,—R^(aa), —N(R^(bb))₂, —C(═O)SR^(aa), —C(═O)R^(aa), —CO₂R^(aa),—C(═O)N(R^(bb))₂, —C(═NR^(bb))R^(aa), —C(═NR^(bb))OR^(aa),—C(═NR^(bb))N(R^(bb))₂, —S(═O)R^(aa), —SO₂R^(aa), —Si(R^(aa))₃,—P(R^(cc))₂, —P(R^(cc))₃ ⁺X⁻, —P(OR^(cc))₂, —P(OR^(cc))₃ ⁺X⁻,—P(═O)(R^(aa))₂, —P(═O)(OR^(cc))₂, and —P(═O)(N(R^(bb))₂)₂, whereinR^(aa), R^(bb), and R^(cc) are as defined herein. Sulfur protectinggroups are well known in the art and include those described in detailin Protecting Groups in Organic Synthesis, T. W. Greene and P. G. M.Wuts, 3^(rd) edition, John Wiley & Sons, 1999, incorporated herein byreference. In certain embodiments, a sulfur protecting group isacetamidomethyl, t-Bu, 3-nitro-2-pyridine sulfenyl, 2-pyridine-sulfenyl,or triphenylmethyl.

A “counterion” or “anionic counterion” is a negatively charged groupassociated with a positively charged group in order to maintainelectronic neutrality. An anionic counterion may be monovalent (i.e.,including one formal negative charge). An anionic counterion may also bemultivalent (i.e., including more than one formal negative charge), suchas divalent or trivalent. Exemplary counterions include halide ions(e.g., F⁻, Cl⁻, Br⁻, I⁻), NO₃ ⁻, ClO₄ ⁻, OH⁻, H₂PO₄ ⁻, HCO₃ ⁻, HSO₄ ⁻,sulfonate ions (e.g., methansulfonate, trifluoromethanesulfonate,p-toluenesulfonate, benzenesulfonate, 10-camphor sulfonate,naphthalene-2-sulfonate, naphthalene-1-sulfonic acid-5-sulfonate,ethan-1-sulfonic acid-2-sulfonate, and the like), carboxylate ions(e.g., acetate, propanoate, benzoate, glycerate, lactate, tartrate,glycolate, gluconate, and the like), BF₄ ⁻, PF₄ ⁻, PF₆ ⁻, AsF₆ ⁻, SbF₆⁻, B[3,5—(CF₃)₂C₆H₃]₄]⁻, B(C₆F₅)₄ ⁻, BPh₄ ⁻, Al(OC(CF₃)₃)₄ ⁻, andcarborane anions (e.g., CB₁₁H₁₂ ⁻ or (HCB₁₁Me₅Br₆)⁻). Exemplarycounterions which may be multivalent include CO₃ ²⁻, HPO₄ ²⁻, PO₄ ³⁻,B₄O₇ ²⁻, SO₄ ²⁻, S₂O₃ ²⁻, carboxylate anions (e.g., tartrate, citrate,fumarate, maleate, malate, malonate, gluconate, succinate, glutarate,adipate, pimelate, suberate, azelate, sebacate, salicylate, phthalates,aspartate, glutamate, and the like), and carboranes.

As used herein, use of the phrase “at least one instance” refers to 1,2, 3, 4, or more instances, but also encompasses a range, e.g., forexample, from 1 to 4, from 1 to 3, from 1 to 2, from 2 to 4, from 2 to3, or from 3 to 4 instances, inclusive.

Other Definitions

As used herein, the term “salt” refers to any and all salts, andencompasses pharmaceutically acceptable salts. The term“pharmaceutically acceptable salt” refers to those salts which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of humans and lower animals without undue toxicity,irritation, allergic response, and the like, and are commensurate with areasonable benefit/risk ratio. Pharmaceutically acceptable salts arewell known in the art. For example, Berge et al. describepharmaceutically acceptable salts in detail in J. PharmaceuticalSciences, 1977, 66, 1-19, incorporated herein by reference.Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from suitable inorganic and organic acids andbases. Examples of pharmaceutically acceptable, nontoxic acid additionsalts are salts of an amino group formed with inorganic acids, such ashydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, andperchloric acid or with organic acids, such as acetic acid, oxalic acid,maleic acid, tartaric acid, citric acid, succinic acid, or malonic acidor by using other methods known in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. Salts derived from appropriate bases include alkali metal,alkaline earth metal, ammonium, and N+(C₁₋₄ alkyl)₄ salts.Representative alkali or alkaline earth metal salts include sodium,lithium, potassium, calcium, magnesium, and the like. Furtherpharmaceutically acceptable salts include, when appropriate, nontoxicammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, lower alkyl sulfonate, and aryl sulfonate. Compounds describedherein are also provided, and can be administered, as a free base.

It is also to be understood that compounds that have the same molecularformula but differ in the nature or sequence of bonding of their atomsor the arrangement of their atoms in space are termed “isomers”. Isomersthat differ in the arrangement of their atoms in space are termed“stereoisomers”.

The terms “composition” and “formulation” are used interchangeably.

A “subject” to which administration is contemplated refers to a human(i.e., male or female of any age group, e.g., pediatric subject (e.g.,infant, child, or adolescent) or adult subject (e.g., young adult,middle-aged adult, or senior adult)) or non-human animal. In certainembodiments, the non-human animal is a mammal (e.g., primate (e.g.,cynomolgus monkey or rhesus monkey), commercially relevant mammal (e.g.,cattle, pig, horse, sheep, goat, cat, or dog), or bird (e.g.,commercially relevant bird, such as chicken, duck, goose, or turkey)).In certain embodiments, the non-human animal is a fish, reptile, oramphibian. The non-human animal may be a male or female at any stage ofdevelopment. The non-human animal may be a transgenic animal orgenetically engineered animal. The term “patient” refers to a humansubject in need of treatment of a disease.

The term “administer,” “administering,” or “administration” refers toimplanting, absorbing, ingesting, injecting, inhaling, or otherwiseintroducing a compound described herein, or a composition thereof, in oron a subject.

The terms “treatment,” “treat,” and “treating” refer to reversing,alleviating, delaying the onset of, or inhibiting the progress of adisease described herein. In some embodiments, treatment may beadministered after one or more signs or symptoms of the disease havedeveloped or have been observed. In other embodiments, treatment may beadministered in the absence of signs or symptoms of the disease. Forexample, treatment may be administered to a susceptible subject prior tothe onset of symptoms. Treatment may also be continued after symptomshave resolved, for example, to delay or prevent recurrence.

An “effective amount” of a compound described herein refers to an amountsufficient to elicit the desired biological response. An effectiveamount of a compound described herein may vary depending on such factorsas the desired biological endpoint, the pharmacokinetics of thecompound, the condition being treated, the mode of administration, andthe age and health of the subject. In certain embodiments, an effectiveamount is a therapeutically effective amount. Alternatively, in aseparate method or use, the invention may be used, where indicated andeffective, as a prophylactic treatment. In certain embodiments, aneffective amount is the amount of a compound described herein in asingle dose. In certain embodiments, an effective amount is the combinedamounts of a compound described herein in multiple doses.

A “therapeutically effective amount” of a compound described herein isan amount sufficient to provide a therapeutic benefit in the treatmentof a condition or to delay or minimize one or more symptoms associatedwith the condition. A therapeutically effective amount of a compoundmeans an amount of therapeutic agent, alone or in combination with othertherapies, which provides a therapeutic benefit in the treatment of thecondition. The term “therapeutically effective amount” can encompass anamount that improves overall therapy, reduces or avoids symptoms, signs,or causes of the condition, and/or enhances the therapeutic efficacy ofanother therapeutic agent. In certain embodiments, a therapeuticallyeffective amount is an amount sufficient for treating in any disease orcondition described.

As used herein, “inhibition”, “inhibiting”, “inhibit” and “inhibitor”,and the like, refer to the ability of a compound to reduce, slow, halt,or prevent the activity of a biological process (e.g., tumor growth). Incertain embodiments, the inhibition is about 45% to 50%. In certainembodiments, the inhibition is about 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99.9%, or100%.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

The present invention is described in detail below with reference toembodiments and the like of the present invention. The inventionprovides compounds (e.g., compounds of Formulae (I), (II), (III), and(IV)), and pharmaceutically acceptable salts or isotopically labeledderivatives thereof, and pharmaceutical compositions thereof. Theinvention also provides methods of treating proliferative diseases(e.g., inhibiting tumor growth and/or treating cancer) in a subjectcomprising administering an effective amount to the subject of acompound or composition provided herein. The compound or composition maybe administered as a monotherapy or in combination with another therapy,as described herein.

In yet another aspect, the present invention provides methods ofpreparing compounds of Formulae (I), (II), (III), and (IV), andsynthetic intermediates useful to that end.

In one aspect, the present invention provides compounds of Formula (I):

and pharmaceutically acceptable salts and isotopically labeledderivatives thereof, wherein:

R^(N1) and R^(N2) are independently hydrogen, optionally substitutedalkyl, optionally substituted acyl, or a nitrogen protecting group,optionally wherein R^(N1) and R^(N2) are joined together with theintervening atoms to form optionally substituted heterocyclyl oroptionally substituted heteroaryl;

R^(P1) is hydrogen, optionally substituted alkyl, optionally substitutedacyl, or an oxygen protecting group;

R^(X) is hydrogen or —OR^(Xa);

R^(Y) is hydrogen or —OR^(Ya);

R^(Xa) and R^(Ya) are independently hydrogen, optionally substitutedalkyl, optionally substituted acyl, or an oxygen protecting group,optionally wherein R^(Xa) and R^(Ya) are joined together with theintervening atoms to form optionally substituted heterocyclyl; and

n is 1 or 2.

In certain embodiments, the compound of formula (I) is Compound (1):

or a pharmaceutically acceptable salt or isotopically labeled derivativethereof. In certain embodiments, the compound of Formula (I) is notCompound (1), or a pharmaceutically acceptable salt or isotopicallylabeled derivative thereof. In certain embodiments, Compound (1) and allpharmaceutically acceptable salts and isotopically labeled derivativesthereof are excluded from the invention.

In certain embodiments, the compound of Formula (I) is of one of thefollowing formulae:

or a pharmaceutically acceptable salt or isotopically labeled derivativethereof.

In certain embodiments, the compound of Formula (I) is of one of thefollowing formulae:

or a pharmaceutically acceptable salt or isotopically labeled derivativethereof.

In certain embodiments, the compound of Formula (I) is selected from thegroup consisting of:

and pharmaceutically acceptable salts and isotopically labeledderivatives thereof.

In another aspect, the present invention provides compounds of Formula(II):

and pharmaceutically acceptable salts and isotopically labeledderivatives thereof, wherein

R^(N1) and R^(N2) are independently hydrogen, optionally substitutedalkyl, optionally substituted acyl, or a nitrogen protecting group,optionally wherein R^(N1) and R^(N2) are joined together with theintervening atoms to form optionally substituted heterocyclyl oroptionally substituted heteroaryl;

R^(P1) and R^(P2) are independently hydrogen, optionally substitutedalkyl, optionally substituted acyl, or an oxygen protecting group;

R^(X) is hydrogen or —OR^(Xa);

R^(Y) is hydrogen or —OR^(Ya);

R^(Xa) and R^(Ya) are independently hydrogen, optionally substitutedalkyl, optionally substituted acyl, or an oxygen protecting group,optionally wherein R^(Xa) and R^(Ya) are joined together with theintervening atoms to form optionally substituted heterocyclyl; and

m is 0 or 1.

In certain embodiments, the compound of Formula (II) is of one of thefollowing formulae:

or a pharmaceutically acceptable salt or isotopically labeled derivativethereof.

In certain embodiments, the compound of Formula (II) is of one of thefollowing formulae:

or a pharmaceutically acceptable salt or isotopically labeled derivativethereof.

In certain embodiments, the compound of Formula (II) is selected fromthe group consisting of

and pharmaceutically acceptable salts and isotopically labeledderivatives thereof.

In another aspect, the present invention provides compounds of Formula(III):

and pharmaceutically acceptable salts and isotopically labeledderivatives thereof, wherein:

R^(O) is hydrogen, optionally substituted alkyl, optionally substitutedacyl, or an oxygen protecting group;

R^(P1) is hydrogen, optionally substituted alkyl, optionally substitutedacyl, or an oxygen protecting group;

R^(X) is hydrogen or —OR^(Xa);

R^(Y) is hydrogen or —OR^(Ya);

R^(Xa) and R^(Ya) are independently hydrogen, optionally substitutedalkyl, optionally substituted acyl, or an oxygen protecting group,optionally wherein R^(Xa) and R^(Ya) are joined together with theintervening atoms to form optionally substituted heterocyclyl; and

n is 1 or 2.

In certain embodiments, the compound of Formula (III) is the following:

or a pharmaceutically acceptable salt thereof. In certain embodiments,the compound of Formula (III) is not (D-6), or a pharmaceuticallyacceptable salt or isotopically labeled derivative thereof. In certainembodiments (D-6), and all pharmaceutically acceptable salts andisotopically labeled derivatives thereof, are excluded from theinvention.

In certain embodiments, the compound of Formula (III) is of one of thefollowing formulae:

or a pharmaceutically acceptable salt or isotopically labeled derivativethereof.

In certain embodiments, the compound of Formula (III) is of one of thefollowing formulae:

or a pharmaceutically acceptable salt or isotopically labeled derivativethereof.

In certain embodiments, the compound of Formula (III) is selected fromthe group consisting of:

or a pharmaceutically acceptable salt or isotopically labeled derivativethereof.

In yet another aspect, the present invention provides compounds ofFormula (IV):

and pharmaceutically acceptable salts and isotopically labeledderivatives thereof, wherein:

R^(O) is hydrogen, optionally substituted alkyl, optionally substitutedacyl, or an oxygen protecting group;

R^(P1) is hydrogen, optionally substituted alkyl, optionally substitutedacyl, or an oxygen protecting group;

R^(X) is hydrogen or —OR^(Xa);

R^(Y) is hydrogen or —OR^(Ya);

R^(Xa) and R^(Ya) are independently hydrogen, optionally substitutedalkyl, optionally substituted acyl, or an oxygen protecting group,optionally wherein R^(Xa) and R^(Ya) are joined together with theintervening atoms to form optionally substituted heterocyclyl; and

m is 0 or 1.

In certain embodiments, the compound of Formula (IV) is of one of thefollowing formulae:

or a pharmaceutically acceptable salt or isotopically labeled derivativethereof.

In certain embodiments, the compound of Formula (IV) is selected fromthe group consisting of

and pharmaceutically acceptable salts and isotopically labeledderivatives thereof.

The compounds provided herein may be optionally be in the form of ahydrate, solvate or polymorph, optionally in a pharmaceuticallyacceptable carrier or excipient. The present invention also providesstereoisomers of any one of the compounds described herein.

Compounds provided herein may exist as a crystal polymorph, and thecompound of the present invention may be in any of single crystal formsor a mixture of two or more crystal forms. Compounds provided herein canbe in an amorphous form, or can be an anhydride or a solvate, such as ahydrate.

The present invention includes isotopically labeled derivatives ofcompounds provided herein, and pharmaceutically acceptable saltsthereof. The isotopically labeled compound is equivalent to compoundsprovided herein, except that one or more atom(s) are replaced by atom(s)having an atomic mass or a mass number different from those usuallyfound in nature. Examples of an isotope that can be incorporated intothe compound of the present invention include isotopes of hydrogen,carbon, nitrogen, oxygen, phosphorus, fluorine, iodine, bromine andchlorine, such as ²H, ³H, ¹¹C, ¹³C, ¹⁴C, ¹⁸F, ³⁵S, ¹²³I, and ¹²⁵I.

The isotopically labeled compound, such as a compound into which aradioactive isotope of, for example, ³H and/or ¹⁴C is incorporated, isuseful for a tissue distribution assay for a medicine and/or a matrix.The isotopes ³H and ¹⁴C are regarded to be useful because these isotopescan be easily prepared and detected. The isotopes ¹¹C and ¹⁸F are usefulin PET (positron emission tomography). The isotope ¹²⁵I is regarded tobe useful in SPECT (single photon emission computed tomography), and canbe useful in brain imaging. Replacement by a heavier isotope such as ²Hcauses, because of its higher metabolic stability, some advantages, in atreatment, of, for example, extension of half-life in vivo or reductionof a necessary dose, and therefore, is regarded useful under givencircumstances. The isotopically labeled compound can be similarlyprepared by using a readily available isotopically labeled reagentinstead of a non-isotopically labeled reagent and by performingprocesses disclosed in schemes and/or examples described below.

Compounds provided herein can be used as a chemical probe for capturinga target protein of a biologically active low molecular weight compound.Specifically, the compound of the present invention can be transformedinto an affinity chromatography probe, a photoaffinity probe or the likeby introducing a labeling group, a linker or the like into a moietyother than a structural moiety indispensable to activity expression ofthe compound by a method described in J. Mass Spectrum. Soc. Jpn. Vol.51, No. 5, 2003, p. 492-498, WO 2007/139149, or the like.

Examples of the labeling group, the linker or the like used in such achemical probe include groups belonging to the following groups (1) to(5). (1) Protein labeling groups such as photoaffinity labeling groups(such as a benzoyl group, a benzophenone group, an azide group, acarbonyl azide group, a diaziridine group, an enone group, a diazo groupand a nitro group), and chemical affinity groups (such as a ketone groupin which an alpha carbon atom is substituted by a halogen atom, acarbamoyl group, an ester group, an alkylthio group, a Michael acceptorof α,β-unsaturated ketone, ester, or the like, and an oxirane group);(2) cleavable linkers such as S—S, O—Si—O, a monosaccharide (such as aglucose group or a galactose group) and a disaccharide (such aslactose), and oligopeptide linkers that can be cleaved by an enzymereaction; (3) fishing tag groups such as biotin and a 3-(4,4-difluoro-5,7-dimethyl-4H-3a,4a-diaza-4-bora-s-indacene-3-yl)propionylgroup; (4) radioactive labeling groups such as ¹²⁵I, ³²P ³H and ¹⁴C;fluorescence labeling groups such as fluorescein, rhodamine, dansyl,umbelliferone, 7-nitrofurazanyl, and a3-(4,4-difluoro-5,7-dimethyl-4H-3a,4a-diaza-4-bora-s-indacene-3-yl)propionyl group; chemiluminescent groups such as luciferin and luminol;and markers capable of detecting heavy metal ions such as lanthanoidmetal ions and radium ions; and (5) groups to be bonded to a solid phasecarrier such as glass beads, a glass bed, a microliter plate, agarosebeads, an agarose bed, polystyrene beads, a polystyrene bed, nylon beadsand a nylon bed.

A probe prepared by introducing, into the compound of the presentinvention, a labeling group or the like selected from theabove-described groups (1) to (5) by the method described in any of theaforementioned literatures or the like can be used as a chemical probefor identifying a marker protein useful for research of a novelpotential drug target.

Examples of a “salt” used herein include salts with inorganic acids,salts with organic acids, and salts with acidic amino acids, and inparticular, pharmaceutically acceptable salts are preferred. Besides, asalt of the compound of the present invention embraces an anhydride of apharmaceutically acceptable salt thereof and a solvate, such as ahydrate, of the pharmaceutically acceptable salt. Preferable examples ofa salt with an inorganic acid include salts with hydrochloric acid,hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and thelike, and preferable examples of a salt with an organic acid includesalts with acetic acid, succinic acid, famaric acid, maleic acid,tartaric acid, citric acid, lactic acid, stearic acid, benzoic acid,methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid,p-toluenesulfonic acid and the like. Preferable examples of a salt withan acidic amino acid include salts with aspartic acid and glutamic acidand the like.

In the case where the compound according to the present invention isobtained as a salt of the compound or a hydrate of the compound, thesalt and the hydrate can be converted to a free body of the compound bya conventional method.

Groups R^(N1), R^(N2), R^(O), R^(P1), R^(P2), R^(X), R^(Y), n, and m

All definitions provided below apply to all compounds of Formulae (I),(II), (II), and (IV) described herein.

As generally defined herein, R^(N1) and R^(N2) are independentlyhydrogen, optionally substituted alkyl, optionally substituted acyl, ora nitrogen protecting group, optionally wherein R^(N1) and R^(N2) arejoined together with the intervening atoms to form optionallysubstituted heterocyclyl or optionally substituted heteroaryl. Incertain embodiments, R^(N1) is hydrogen. In certain embodiments, R^(N1)is optionally substituted alkyl. In certain embodiments, R^(N1) isoptionally substituted acyl. In certain embodiments, R^(N1) is anitrogen protecting group. In certain embodiments, R^(N1) is an esterprotecting group. In certain embodiments, R^(N1) is an alloc protectinggroup. “Alloc” is of the following formula:

In certain embodiments, R^(N2) is hydrogen. In certain embodiments,R^(N2) is optionally substituted alkyl. In certain embodiments, R^(N2)is optionally substituted acyl. In certain embodiments, R^(N2) is anitrogen protecting group. In certain embodiments, R^(N2) is an esterprotecting group. In certain embodiments, R^(N2) is an alloc protectinggroup.

In certain embodiments, R^(N1) and R^(N2) are both hydrogen. In certainembodiments, R^(N1) is hydrogen; and R^(N2) is a nitrogen protectinggroup. In certain embodiments, R^(N1) is hydrogen; and R^(N2) is alloc.In certain embodiments, R^(N1) and R^(N2) are joined together with theintervening atoms to form optionally substituted heterocyclyl. Incertain embodiments, R^(N1) and R^(N2) are joined together with theintervening atoms to form optionally substituted heteroaryl.

As generally defined herein, R^(P1) is hydrogen, optionally substitutedalkyl, optionally substituted acyl, or an oxygen protecting group. Incertain embodiments, R^(P1) is hydrogen. In certain embodiments, R^(P1)is optionally substituted alkyl. In certain embodiments, R^(P1) isoptionally substituted acyl. In certain embodiments, R^(P1) is an oxygenprotecting group. In certain embodiments, R^(P1) is silyl (e.g.,trialkylsilyl). In certain embodiments, R^(P1) istert-butyldimethylsilyl (TBS or TBDMS).

As generally defined herein, n is 1 or 2. In certain embodiments, nis 1. In certain embodiments, n is 2.

As generally defined herein, m is 0 or 1. In certain embodiments, m is0. In certain embodiments, m is 1.

As generally defined herein, R^(X) is hydrogen or —OR^(Xa). In certainembodiments, R^(X) is hydrogen. In certain embodiments, R^(X)—OR^(Xa).In certain embodiments, R^(X) is —OH.

As generally defined herein, R^(Xa) is hydrogen, optionally substitutedalkyl, optionally substituted acyl, or an oxygen protecting group. Incertain embodiments, R^(Xa) is hydrogen. In certain embodiments, R^(Xa)is optionally substituted alkyl. In certain embodiments, R^(Xa) isoptionally substituted acyl. In certain embodiments, R^(Xa) is an oxygenprotecting group.

As generally defined herein, R^(Y) is hydrogen or —OR^(Ya). In certainembodiments, R^(Y) is hydrogen. In certain embodiments, R^(Y) is—OR^(Ya). In certain embodiments, R^(Y) is —OH.

As generally defined herein, R^(Ya) is hydrogen, optionally substitutedalkyl, optionally substituted acyl, or an oxygen protecting group. Incertain embodiments, R^(Ya) is hydrogen. In certain embodiments, R^(Ya)is optionally substituted alkyl. In certain embodiments, R^(Ya) isoptionally substituted acyl. In certain embodiments, R^(Ya) is an oxygenprotecting group.

In certain embodiments, R^(Xa) and R^(Ya) are joined together with theintervening atoms to form optionally substituted heterocyclyl (e.g.,optionally substituted 5-membered heterocyclyl).

In certain embodiments, R^(X) and R^(Y) are hydrogen. In certainembodiments, R^(X) is —OR^(Xa); and R^(Y) is —OR^(Ya). In certainembodiments, R^(X) and R^(Y) are —OH. In certain embodiments, R^(X) ishydrogen; and R^(Y) is —OR^(Ya). In certain embodiments, R^(X) ishydrogen; and R^(Y) is —OH.

As generally defined herein, R¹² is hydrogen, optionally substitutedalkyl, optionally substituted acyl, or an oxygen protecting group. Incertain embodiments, R¹² is hydrogen. In certain embodiments, R^(P2) isoptionally substituted alkyl. In certain embodiments, R^(P2) isoptionally substituted acyl. In certain embodiments, R^(P2) is an oxygenprotecting group. In certain embodiments, R^(P2) is silyl (e.g.,trialkylsilyl). In certain embodiments, R^(P2) istert-butyldimethylsilyl (TBS or TBDMS).

As generally defined herein, R^(O) is hydrogen, optionally substitutedalkyl, optionally substituted acyl, or an oxygen protecting group. Incertain embodiments, R^(O) is optionally substituted alkyl. In certainembodiments, R^(O) is optionally substituted C₁₋₆ alkyl. In certainembodiments, R^(O) is unsubstituted C₁₋₆ alkyl. In certain embodiments,R^(O) is optionally substituted C₁₋₃ alkyl. In certain embodiments,R^(O) is unsubstituted C₁₋₃ alkyl. In certain embodiments, R^(O) ismethyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, sec-butyl, ortert-butyl. In certain embodiments, R^(O) is methyl. In certainembodiments, R^(O) is optionally substituted acyl. In certainembodiments, R^(O) is an oxygen protecting group.

Pharmaceutical Compositions, Kits, and Administration

The present invention provides pharmaceutical compositions comprising acompound of Formula (I), (II), (III), or (IV), or a pharmaceuticallyacceptable salt or isotopically labeled derivative thereof, and apharmaceutically acceptable excipient. In certain embodiments, thecompound described herein, or pharmaceutically acceptable salt orisotopically labeled derivative thereof, is provided in an effectiveamount in the pharmaceutical composition (e.g., a therapeuticallyeffective amount).

Pharmaceutical compositions described herein can be prepared by anymethod known in the art of pharmacology. In general, such preparatorymethods include bringing a compound provided herein (i.e., the “activeingredient”) into association with a carrier or excipient, and/or one ormore other accessory ingredients, and then, if necessary and/ordesirable, shaping, and/or packaging the product into a desired single-or multi-dose unit.

A pharmaceutical composition of the invention could be preparedaccording to the known method such as a method described in the generalrules for preparations of the Japanese Pharmacopoeia, 16^(th) edition,the United States Pharmacopoeia, and the European Pharmacopoeia, 9^(th)edition. A pharmaceutical composition of the invention could beadministered to patients appropriately depending on the dosage form.

Pharmaceutical compositions can be prepared, packaged, and/or sold inbulk, as a single unit dose, and/or as a plurality of single unit doses.A “unit dose” is a discrete amount of the pharmaceutical compositioncomprising a predetermined amount of the active ingredient. The amountof the active ingredient is generally equal to the dosage of the activeingredient which would be administered to a subject and/or a convenientfraction of such a dosage, such as one-half or one-third of such adosage.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition described herein will vary, depending uponthe identity, size, and/or condition of the subject treated and furtherdepending upon the route by which the composition is to be administered.The composition may comprise between 0.1% and 100% (w/w) activeingredient.

Pharmaceutically acceptable excipients used in the manufacture ofprovided pharmaceutical compositions include inert diluents, dispersingand/or granulating agents, surface active agents and/or emulsifiers,disintegrating agents, binding agents, preservatives, buffering agents,lubricating agents, and/or oils. Excipients such as cocoa butter andsuppository waxes, coloring agents, coating agents, sweetening,flavoring, and perfuming agents may also be present in the composition.

The compound provided herein are typically formulated in dosage unitform for ease of administration and uniformity of dosage. It will beunderstood, however, that the total daily usage of the compositionsdescribed herein will be decided by a physician within the scope ofsound medical judgment. The specific therapeutically effective doselevel for any particular subject or organism will depend upon a varietyof factors including the disease being treated and the severity of thedisorder; the activity of the specific active ingredient employed; thespecific composition employed; the age, body weight, general health,sex, and diet of the subject; the time of administration, route ofadministration, and rate of excretion of the specific active ingredientemployed; the duration of the treatment; drugs used in combination orcoincidental with the specific active ingredient employed; and likefactors well known in the medical arts.

The compounds of the present invention and compositions thereof providedherein can be administered by any route, including enteral (e.g., oral),parenteral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, subcutaneous, intraventricular, transdermal, interdermal,rectal, intravaginal, intraperitoneal, topical (as by powders,ointments, creams, and/or drops), mucosal, nasal, bucal, sublingual; byintratracheal instillation, bronchial instillation, and/or inhalation;and/or as an oral spray, nasal spray, and/or aerosol. Specificallycontemplated routes are oral administration, intravenous administration(e.g., systemic intravenous injection), regional administration viablood and/or lymph supply, and/or direct administration to an affectedsite. In general, the most appropriate route of administration willdepend upon a variety of factors including the nature of the agent(e.g., its stability in the environment of the gastrointestinal tract),and/or the condition of the subject (e.g., whether the subject is ableto tolerate oral administration).

The exact amount of a compound provided herein required to achieve aneffective amount will vary from subject to subject, depending, forexample, on species, age, and general condition of a subject, severityof the side effects or disorder, identity of the particular compound,mode of administration, and the like. An effective amount may beincluded in a single dose (e.g., single oral dose) or multiple doses(e.g., multiple oral doses). In certain embodiments, when multiple dosesare administered to a subject or applied to a tissue or cell, any twodoses of the multiple doses include different or substantially the sameamounts of a compound described herein. In certain embodiments, whenmultiple doses are administered to a subject or applied to a tissue orcell, the frequency of administering the multiple doses to the subjector applying the multiple doses to the tissue or cell may be, innon-limiting examples, three doses a day, two doses a day, one dose aday, one dose every other day, one dose every third day, one dose everyweek, one dose every two weeks, one dose every three weeks, or one doseevery four weeks, or even slow dose controlled delivery over a selectedperiod of time using a drug delivery device. In certain embodiments, thefrequency of administering the multiple doses to the subject or applyingthe multiple doses to the tissue or cell is one dose per day. In certainembodiments, the frequency of administering the multiple doses to thesubject or applying the multiple doses to the tissue or cell is twodoses per day. In certain embodiments, the frequency of administeringthe multiple doses to the subject or applying the multiple doses to thetissue or cell is three doses per day. In certain embodiments, whenmultiple doses are administered to a subject or applied to a tissue orcell, the duration between the first dose and last dose of the multipledoses is about or at least one day, two days, four days, one week, twoweeks, three weeks, one month, two months, three months, four months,six months, nine months, one year, two years, three years, four years,five years, seven years, ten years, fifteen years, twenty years, or thelifetime of the subject, tissue, or cell. In certain embodiments, theduration between the first dose and last dose of the multiple doses isabout or at least three months, six months, or one year. In certainembodiments, the duration between the first dose and last dose of themultiple doses is the lifetime of the subject, tissue, or cell. Incertain embodiments, a dose (e.g., a single dose, or any dose ofmultiple doses) described herein includes independently between 0.001mg/kg and 0.01 mg/kg, between 0.01 mg/kg and 0.1 mg/kg, between 0.1mg/kg and 1 mg/kg, inclusive of a compound provided herein. Examples aredosage forms with at least about 0.05, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5,4, 4.5, 5, 10, 5, 20, 25, or 50 mg of active compound, or its salt in adosage form.

In certain embodiments, a dose (e.g., a single dose, or any of multipledoses) described herein includes independently between 1.0 μg/m² and 1.0mg/m², inclusive, of a compound provided herein (e.g., Compound (1) or apharmaceutically acceptable salt thereof). In certain embodiments, adose (e.g., a single dose, or any dose of multiple doses) describedherein includes independently between 1.0 μg/m² and 100.0 μg/m², between1.0 μg/m² and 50 μg/m², between 10 μg/m² and 50 μg/m², or between 10μg/m² and 30 μg/m², inclusive, of a compound provided herein (e.g.,Compound (1) or a pharmaceutically acceptable salt thereof). Examplesare dosage forms with approximately 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, and 40 μg of a compound provided herein (e.g., Compound (1)or a pharmaceutically acceptable salt thereof).

In certain embodiments, a compound provided herein (e.g., Compound (1)or a pharmaceutically acceptable salt thereof) is dosed approximatelyonce every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days. In certainembodiments, a compound provided herein (e.g., Compound (1) or apharmaceutically acceptable salt thereof) is dosed approximately twice amonth. In certain embodiments, a compound provided herein (e.g.,Compound (1) or a pharmaceutically acceptable salt thereof) is dosedapproximately once every 15 days for the duration of the treatment. Incertain embodiments, a compound provided herein (e.g., Compound (1) or apharmaceutically acceptable salt thereof) is dosed on day 1 and day 15of a 28-day cycle. In certain embodiments, a compound provided herein(e.g., Compound (1) or a pharmaceutically acceptable salt thereof) isdosed at approximately 25 μg/m² on approximately day 1 and approximatelyday 15 of an approximately 28-day cycle. In certain embodiments, acompound provided herein (e.g., Compound (1) or a pharmaceuticallyacceptable salt thereof) is dosed at 25 μg/m² at on day 1 and day 15 ofa 28-day cycle. The number of cycles will be decided by a physician.

Dose ranges as described herein provide guidance for the administrationof provided pharmaceutical compositions to an adult. The amount to beadministered to, for example, a child or an adolescent can be determinedby a medical practitioner or person skilled in the art and can be loweror the same as that administered to an adult.

Also encompassed by the disclosure are kits (e.g., pharmaceuticalpacks). The kits provided may comprise a pharmaceutical composition orcompound provided herein and a container (e.g., a vial, ampule, bottle,syringe, and/or dispenser package, or other suitable container). In someembodiments, provided kits may optionally further include a secondcontainer comprising a pharmaceutical excipient for dilution orsuspension of a pharmaceutical composition or compound provided herein.In some embodiments, the pharmaceutical composition or compound providedin the first container and the second container are combined to form oneunit dosage form. A kit described herein may include one or moreadditional pharmaceutical agents described herein as a separatecomposition.

Methods of Treatment and Uses

As shown herein, compounds of Formulae (I), (II), (III), and (IV) havesignificant tumor vascular remodeling effects and anti-CAF activity, andtherefore, have potential use for the treatment of proliferativediseases (e.g., treatment of cancer and/or the inhibition of tumorgrowth).

Provided herein is a method of treating a proliferative disease in asubject, the method comprising administering to the subject an effectiveamount of a compound provided herein, or a pharmaceutically acceptablesalt or isotopically labeled derivative thereof, or a pharmaceuticalcomposition thereof. The present invention also provides a compounddescribed herein, or a pharmaceutically acceptable salt or isotopicallylabeled derivative thereof, or a pharmaceutical composition thereof, foruse in treating a proliferative disease in a subject. The presentinvention also provides the use of a compound provided herein, or apharmaceutically acceptable salt or isotopically labeled derivativethereof, or a pharmaceutical composition thereof, for the manufacture ofa medicament for the treating a proliferative disease. Examples ofproliferative diseases are described herein.

Provided herein is a method of treating cancer in a subject, the methodcomprising administering to the subject an effective amount of acompound provided herein, or a pharmaceutically acceptable salt orisotopically labeled derivative thereof, or a pharmaceutical compositionthereof. The present invention also provides a compound describedherein, or a pharmaceutically acceptable salt or isotopically labeledderivative thereof, or a pharmaceutical composition thereof, for use intreating cancer in a subject. The present invention also provides theuse of a compound provided herein, or a pharmaceutically acceptable saltor isotopically labeled derivative thereof, or a pharmaceuticalcomposition thereof, for the manufacture of a medicament for thetreating cancer.

Also provided herein is a method of inhibiting tumor growth in asubject, the method comprising administering to the subject a compoundprovided herein, or a pharmaceutically acceptable salt or isotopicallylabeled derivative thereof, or a pharmaceutical composition thereof.Also provided herein is a compound described herein, or apharmaceutically acceptable salt or isotopically labeled derivativethereof, or a pharmaceutical composition thereof, for use in inhibitingtumor growth in a subject. The present invention also provides the useof a compound provided herein, or a pharmaceutically acceptable salt orisotopically labeled derivative thereof, or a pharmaceutical compositionthereof, for the manufacture of a medicament for inhibiting tumorgrowth.

A “proliferative disease” refers to a disease that occurs due toabnormal growth or extension by the multiplication of cells (Walker,Cambridge Dictionary of Biology; Cambridge University Press: Cambridge,UK, 1990). A proliferative disease may be associated with: 1) thepathological proliferation of normally quiescent cells; 2) thepathological migration of cells from their normal location (e.g.,metastasis of neoplastic cells); 3) the pathological expression ofproteolytic enzymes such as the matrix metalloproteinases (e.g.,collagenases, gelatinases, and elastases); or 4) the pathologicalangiogenesis as in proliferative retinopathy and tumor metastasis.Exemplary proliferative diseases include cancers (i.e., “malignantneoplasms”), benign neoplasms, angiogenesis, inflammatory diseases, andautoimmune diseases.

The term “angiogenesis” refers to the physiological process throughwhich new blood vessels form from pre-existing vessels. Angiogenesis isdistinct from vasculogenesis, which is the de novo formation ofendothelial cells from mesoderm cell precursors. The first vessels in adeveloping embryo form through vasculogenesis, after which angiogenesisis responsible for most blood vessel growth during normal or abnormaldevelopment.

Angiogenesis is a vital process in growth and development, as well as inwound healing and in the formation of granulation tissue. However,angiogenesis is also a fundamental step in the transition of tumors froma benign state to a malignant one, leading to the use of angiogenesisinhibitors in the treatment of cancer. Angiogenesis may be chemicallystimulated by angiogenic proteins, such as growth factors (e.g., VEGF).“Pathological angiogenesis” refers to abnormal (e.g., excessive orinsufficient) angiogenesis that amounts to and/or is associated with adisease.

The terms “neoplasm” and “tumor” are used herein interchangeably andrefer to an abnormal mass of tissue wherein the growth of the masssurpasses and is not coordinated with the growth of a normal tissue. Aneoplasm or tumor may be “benign” or “malignant,” depending on thefollowing characteristics: degree of cellular differentiation (includingmorphology and functionality), rate of growth, local invasion, andmetastasis. A “benign neoplasm” is generally well differentiated, hascharacteristically slower growth than a malignant neoplasm, and remainslocalized to the site of origin. In addition, a benign neoplasm does nothave the capacity to infiltrate, invade, or metastasize to distantsites.

Exemplary benign neoplasms include, but are not limited to, lipoma,chondroma, adenomas, acrochordon, senile angiomas, seborrheic keratoses,lentigos, and sebaceous hyperplasias. In some cases, certain “benign”tumors may later give rise to malignant neoplasms, which may result fromadditional genetic changes in a subpopulation of the tumor's neoplasticcells, and these tumors are referred to as “pre-malignant neoplasms.” Anexemplary pre-malignant neoplasm is a teratoma. In contrast, a“malignant neoplasm” is generally poorly differentiated (anaplasia) andhas characteristically rapid growth accompanied by progressiveinfiltration, invasion, and destruction of the surrounding tissue.Furthermore, a malignant neoplasm generally has the capacity tometastasize to distant sites. The term “metastasis,” “metastatic,” or“metastasize” refers to the spread or migration of cancerous cells froma primary or original tumor to another organ or tissue and is typicallyidentifiable by the presence of a “secondary tumor” or “secondary cellmass” of the tissue type of the primary or original tumor and not ofthat of the organ or tissue in which the secondary (metastatic) tumor islocated.

In certain embodiments, the disease to be treated is cancer. The term“cancer” refers to a class of diseases characterized by the developmentof abnormal cells that proliferate uncontrollably and have the abilityto infiltrate and destroy normal body tissues.

In certain embodiments, the cancer is head and neck cancer (e.g.,squamous cell carcinoma of the head and neck (SCCHN), adenoid cysticcarcinoma).

In certain embodiments, the cancer is oral cancer (e.g., buccal cavitycancer, lip cancer, tongue cancer, mouth cancer, pharynx cancer,hypopharynx cancer (e.g., hypopharyngeal carcinoma), throat cancer(e.g., laryngeal cancer, pharyngeal cancer, nasopharyngeal cancer,oropharyngeal cancer), salivary gland cancer).

In certain embodiments, the cancer is esophageal cancer (e.g.,esophageal squamous cell carcinoma, esophageal adenocarcinoma, Barrett'sadenocarcinoma, esophageal leiomyosarcoma).

In certain embodiments, the cancer is gastrointestinal cancer (e.g.,anal cancer, colorectal cancer (e.g., colon cancer, rectal cancer,colorectal adenocarcinoma), gall bladder cancer, gastric cancer (e.g.,stomach cancer (e.g., stomach adenocarcinoma)), gastrointestinal stromaltumor (GIST), small bowel cancer (e.g., appendix cancer, small bowelcarcinoma, e.g., small bowel adenocarcinoma), small intestine cancer,large bowel cancer, large intestine cancer).

In certain embodiments, the cancer is cardiovascular cancer (e.g.,primary cardiac tumors, angiosarcoma (e.g., lymphangiosarcoma,lymphangioendotheliosarcoma, hemangiosarcoma), endotheliosarcoma (e.g.,Kaposi's sarcoma, multiple idiopathic hemorrhagic sarcoma), cardiacmyxoma, cardiac rhabdomyoma).

In certain embodiments, the cancer is lung cancer (e.g., bronchus cancer(e.g., bronchogenic carcinoma, bronchial adenoma), alveolar carcinoma,mesothelioma, small cell lung cancer (SCLC), non-small cell lung cancer(NSCLC), lung adenocarcinoma, chondromatous hamartoma, papillaryadenocarcinoma).

In certain embodiments, the cancer is a genitourinary cancer (e.g.,bladder cancer (e.g., urothelial carcinoma), urethral cancer, kidneycancer (e.g., nephroblastoma a.k.a.

Wilms' tumor, renal cell carcinoma), testicular cancer (e.g., seminoma,testicular embryonal carcinoma), germ cell cancer, prostate cancer(e.g., prostate adenocarcinoma), penile cancer (e.g., Paget's disease ofthe penis and scrotum)).

In certain embodiments, the cancer is a gynecological cancer (e.g.,breast cancer (e.g., adenocarcinoma of the breast, papillary carcinomaof the breast, mammary cancer, medullary carcinoma of the breast, triplenegative breast cancer, HER-2 positive breast cancer, HER2-negativebreast cancer), endometrial cancer (e.g., uterine cancer (e.g., uterinesarcoma, choriocarcinoma), endometrial carcinoma), cervical cancer(e.g., cervical adenocarcinoma), ovarian cancer (e.g.,cystadenocarcinoma, ovarian embryonal carcinoma, ovarianadenocarcinoma), germ cell cancer, vulvar cancer (e.g., Paget's diseaseof the vulva) vaginal cancer, fallopian tube cancer).

In certain embodiments, the cancer is a hematopoietic cancer (e.g.,leukemia (e.g., acute lymphocytic leukemia (ALL) (e.g., B-cell ALL,T-cell ALL), acute myelocytic leukemia (AML) (e.g., B-cell AML, T-cellAML), chronic myelocytic leukemia (CML) (e.g., B-cell CML, T-cell CML),chronic lymphocytic leukemia (CLL) (e.g., B-cell CLL, T-cell CLL));lymphoma (e.g., Hodgkin lymphoma (HL) (e.g., B-cell HL, T-cell HL)),non-Hodgkin lymphoma (NHL) (e.g., B-cell NHL such as diffuse large celllymphoma (DLCL) (e.g., diffuse large B-cell lymphoma)), follicularlymphoma, chronic lymphocytic leukemia/small lymphocytic lymphoma(CLL/SLL), mantle cell lymphoma (MCL), marginal zone B-cell lymphomas(e.g., mucosa-associated lymphoid tissue (MALT) lymphomads, nodalmarginal zone B-cell lymphoma, splenic marginal zone B-cell lymphoma),primary mediastinal B-cell lymphoma, Burkitt lymphoma, lymphoplasmacyticlymphoma (i.e., Waldenström's macroglobulinemia), hairy cell leukemia(HCL), immunoblastic large cell lymphoma, precursor B-lymphoblasticlymphoma and primary central nervous system (CNS) lymphoma, T-cell NHLsuch as precursor T-lymphoblastic lymphoma/leukemia, peripheral T-celllymphoma (PTCL) (e.g., cutaneous T-cell lymphoma (CTCL) (e.g., mycosisfungoides, Sezary syndrome)), angioimmunoblastic T-cell lymphoma,extranodal natural killer T-cell lymphoma, enteropathy type T-celllymphoma, subcutaneous panniculitis-like T-cell lymphoma, anaplasticlarge cell lymphoma); heavy chain disease (e.g., alpha chain disease,gamma chain disease, mu chain disease); a myeloproliferative disorder(MPD) (e.g., polycythemia vera (PV), essential thrombocytosis (ET),agnogenic myeloid metaplasia (AMM) a.k.a. myelofibrosis (MF), chronicidiopathic myelofibrosis, chronic myelocytic leukemia (CML), chronicneutrophilic leukemia (CNL), hypereosinophilic syndrome (HES)); multiplemyeloma (MM); plasma cell neoplasia; familiar hypereosinophilia;inflammatory myofibroblastic tumors; immunocytic amyloidosis). Incertain embodiments, the cancer is leukemia. In certain embodiments, thecancer is acute lymphoblastic leukemia (ALL). In certain embodiments,the cancer is early T-cell precursor (ETP)-acute lymphoblastic leukemia(ALL).

In certain embodiments, the cancer is liver cancer (e.g., hepatocellularcancer (HCC) (e.g., hepatocellular carcinoma, hepatoblastoma,hepatocellular adenoma), malignant hepatoma, hemangiomas, biliary cancer(e.g., cholangiocarcinoma)).

In certain embodiments, the cancer is musculoskeletal cancer (e.g., bonecancer (e.g., osteosarcoma, osteoid osteoma, malignant fibroushistiocytoma, Ewing's sarcoma, chordoma, malignant giant cell tumorchordoma, chondrosarcoma osteochondroma, benign chondroma,chondroblastoma chondromyxofibroma, myelodysplastic syndrome (MD)),muscle cancer (e.g., rhabdomyosarcoma, rhabdomyoma), connective tissuecancer, synovioma).

In certain embodiments, the cancer is a nervous system cancer (e.g.,brain cancer (e.g., astrocytoma, medulloblastoma, glioma (e.g.,astrocytoma, oligodendroglioma), glioblastomas, glioblastoma multiform,medulloblastoma, ependymoma, germinoma (i.e., pinealoma),oligodendroglioma, schwannoma, retinoblastoma, congenital tumors,craniopharyngioma), spinal cord cancer, neurofibroma (e.g.,neurofibromatosis (NF) type 1 or type 2, schwannomatosis),neuroblastoma, primitive neuroectodermal tumors (PNT), meningeal cancer(e.g., meningioma, meningiosarcoma, gliomatosis), skull cancer, acousticneuroma, ependymoma, hemangioblastoma, ocular cancer (e.g., intraocularmelanoma, retinoblastoma)). In certain embodiments, the disease to betreated is a brain tumor. In certain embodiments, the disease ispleomorphic xenoanthrocytoma (PXA). In certain embodiments, the diseaseis pediatric pleomorphic xenoanthrocytoma (PXA).

In certain embodiments, the cancer is selected from endocrine/exocrinecancers (e.g., thyroid cancer (e.g., papillary thyroid carcinoma,follicular thyroid carcinoma; medullary thyroid carcinoma, multipleendocrine neoplasia type 2A, multiple endocrine neoplasia type 2B,familial medullary thyroid cancer, pheochromocytoma, paraganglioma),pancreatic cancer (e.g., pancreatic andenocarcinoma, intraductalpapillary mucinous neoplasm (IPMN), Islet cell tumors, ductaladenocarcinoma, insulinoma, glucagonoma, vipoma), adrenal gland cancer,neuroendocrine cancer (e.g., gastroenteropancreatic neuroendoctrinetumor (GEP-NET), carcinoid tumor), sebaceous gland carcinoma, sweatgland carcinoma). In certain embodiments, the cancer is sweat glandcancer (e.g., sweat gland carcinoma)

In certain embodiments, the cancer is skin cancer (e.g., squamous cellcarcinoma (SCC), keratoacanthoma (KA), melanoma, basal cell carcinoma(BCC), dermatofribroma).

In certain embodiments, the cancer is a soft tissue cancer (e.g.,intraepithelial neoplasms, epithelial carcinomas, epithelial sarcomas,adenocarcinomas, adenomas, fibrosarcomas, fibromas, liposarcomas,lipomas, myxomas, teratomas).

In certain embodiments, the cancer is a rare cancer. The term “rarecancer” refers to cancers that occur in a relatively small number ofpatients. Rare cancers include, but are not limited to, sarcomas (e.g.,soft tissue sarcoma, liposarcoma, uterine sarcoma, leiomyosarcoma,myxofibrosarcoma, osteosarcoma, angiosarcoma, Ewing's sarcoma, synovialsarcoma, rhabdomyosarcoma, intimal sarcoma), malignant lymphomas, thymiccancer (e.g., thymomas), mesothelioma, gastrointestinal stromal tumors(GISTs), neuroendocrine cancer, eye cancer, brain tumors, bone softtissue tumors, skin cancer, and germ cell tumors.

Examples of proliferative diseases, cancers, and tumors are providedherein. In certain embodiments of the methods provided herein, thecancer is head and neck cancer (e.g., squamous cell carcinoma of thehead and neck, adenoid cystic carcinoma, oral cancer, throat cancer,salivary gland cancer, tongue cancer). In certain embodiments, thecancer is breast cancer (e.g., HER2-positive breast cancer,HER2-negative breast cancer, triple negative breast cancer). In certainembodiments, the cancer is colorectal cancer (e.g., colon carcinoma). Incertain embodiments, the cancer is esophageal cancer (e.g., esophagealadenocarcinoma). In certain embodiments, the cancer is uterine cancer(e.g., uterine sarcoma). In certain embodiments, the cancer is ovariancancer. In certain embodiments, the cancer is a sarcoma (e.g., uterinesarcoma, fibrosarcoma, angiosarcoma, synovial sarcoma, soft tissuesarcoma, intimal sarcoma). In certain embodiments, the cancer is gastriccancer. In certain embodiments, the cancer is lung cancer (e.g.,non-small cell lung cancer). In certain embodiments, the cancer isbladder cancer (e.g., urothelial carcinoma). In certain embodiments, thecancer is endometrial cancer. In certain embodiments, the cancer issmall bowel cancer (e.g., small bowel carcinoma, e.g., small boweladenocarcinoma). In certain embodiments, the cancer is sweat glandcancer (e.g., sweat gland carcinoma). In certain embodiments, the canceris a rare cancer.

Combination Therapy

Besides administration as monotherapy, compounds provided herein (e.g.,compounds of Formulae (I), (II), (III), and (IV)) can be administered incombination with other therapeutic agents or treatment modalities. Incertain embodiments, the compound is administered in combination with ananti-cancer agent. “Anti-cancer agents” encompass biotherapeuticanti-cancer agents as well as chemotherapeutic agents.

Exemplary biotherapeutic anti-cancer agents include, but are not limitedto, interferons, cytokines (e.g., tumor necrosis factor, interferon α,interferon γ), vaccines, hematopoietic growth factors, monoclonalserotherapy, immunostimulants and/or immunodulatory agents (e.g., IL-1,2, 4, 6, or 12), immune cell growth factors (e.g., GM-CSF), andantibodies (e.g., Herceptin (trastuzumab), T-DM1, AVASTIN (bevacizumab),ERBITUX (cetuximab), Vectibix (panitumumab), Rituxan (rituximab), Bexxar(tositumomab), and other antibodies described above.

Exemplary chemotherapeutic agents include, but are not limited to,anti-estrogens (e.g. tamoxifen, raloxifene, and megestrol), LHRHagonists (e.g. goscrclin and leuprolide), anti-androgens (e.g. flutamideand bicalutamide), photodynamic therapies (e.g. vertoporfin (BPD-MA),phthalocyanine, photosensitizer Pc4, and demethoxy-hypocrellin A(2BA-2-DMHA)), nitrogen mustards (e.g. cyclophosphamide, ifosfamide,trofosfamide, chlorambucil, estramustine, and melphalan), nitrosoureas(e.g. carmustine (BCNU) and lomustine (CCNU)), alkylsulphonates (e.g.busulfan and treosulfan), triazenes (e.g. dacarbazine, temozolomide),platinum containing compounds (e.g. cisplatin, carboplatin,oxaliplatin), vinca alkaloids (e.g. vincristine, vinblastine, vindesine,and vinorelbine), taxoids (e.g. paclitaxel or a paclitaxel equivalentsuch as nanoparticle albumin-bound paclitaxel (Abraxane),docosahexaenoic acid bound-paclitaxel (DHA-paclitaxel, Taxoprexin),polyglutamate bound-paclitaxel (PG-paclitaxel, paclitaxel poliglumex,CT-2103, XYOTAX), the tumor-activated prodrug (TAP) ANG1005 (Angiopep-2bound to three molecules of paclitaxel), paclitaxel-EC-1 (paclitaxelbound to the erbB2-recognizing peptide EC-1), and glucose-conjugatedpaclitaxel, e.g., 2′-paclitaxel methyl 2-glucopyranosyl succinate;docetaxel, taxol), epipodophyllins (e.g. etoposide, etoposide phosphate,teniposide, topotecan, 9-aminocamptothecin, camptoirinotecan,irinotecan, crisnatol, mitomycin C), anti-metabolites, DHFR inhibitors(e.g. methotrexate, dichloromethotrexate, trimetrexate, edatrexate), IMPdehydrogenase inhibitors (e.g. mycophenolic acid, tiazofurin, ribavirin,and EICAR), ribonuclotide reductase inhibitors (e.g. hydroxyurea anddeferoxamine), uracil analogs (e.g. 5-fluorouracil (5-FU), floxuridine,doxifluridine, ratitrexed, tegafur-uracil, capecitabine), cytosineanalogs (e.g. cytarabine (ara C), cytosine arabinoside, andfludarabine), purine analogs (e.g. mercaptopurine and Thioguanine),Vitamin D3 analogs (e.g. EB 1089, CB 1093, and KH 1060), isoprenylationinhibitors (e.g. lovastatin), dopaminergic neurotoxins (e.g.1-methyl-4-phenylpyridinium ion), cell cycle inhibitors (e.g.staurosporine), actinomycin (e.g. actinomycin D, dactinomycin),bleomycin (e.g. bleomycin A2, bleomycin B2, peplomycin), anthracycline(e.g. daunorubicin, doxorubicin, pegylated liposomal doxorubicin,idarubicin, epirubicin, pirarubicin, zorubicin, mitoxantrone), MDRinhibitors (e.g. verapamil), Ca²⁺ ATPase inhibitors (e.g. thapsigargin),imatinib, thalidomide, lenalidomide, tyrosine kinase inhibitors (e.g.,axitinib (AG013736), bosutinib (SKI-606), cediranib (RECENTIN™,AZD2171), dasatinib (SPRYCEL®, BMS-354825), erlotinib (TARCEVA®),gefitinib (IRESSA®), imatinib (Gleevec®, CGP57148B, STI-571), lapatinib(TYKERB®, TYVERB®), lestaurtinib (CEP-701), neratinib (HKI-272),nilotinib (TASIGNA®), semaxanib (semaxinib, SU5416), sunitinib (SUTENT®,SU11248), toceranib (PALLADIA®), vandetanib (ZACTIMA®, ZD6474),vatalanib (PTK787, PTK/ZK), trastuzumab (HERCEPTIN®), bevacizumab(AVASTIN®), rituximab (RITUXAN®), cetuximab (ERBITUX®), panitumumab(VECTIBIX®), ranibizumab (Lucentis®), nilotinib (TASIGNA®), sorafenib(NEXAVAR®), everolimus (AFINITOR®), alemtuzumab (CAMPATH®), gemtuzumabozogamicin (MYLOTARG®), temsirolimus (TORISEL®), ENMD-2076, PC1-32765,AC220, dovitinib lactate (TK1258, CHIR-258), BIBW 2992 (TOVOK™), SGX523,PF-04217903, PF-02341066, PF-299804, BMS-777607, ABT-869, MP470, BIBF1120 (VARGATEF®), AP24534, JNJ-26483327, MGCD265, DCC-2036, BMS-690154,CEP-11981, tivozanib (AV-951), OSI-930, MM-121, XL-184, XL-647, and/orXL228), proteasome inhibitors (e.g., bortezomib (Velcade)), mTORinhibitors (e.g., rapamycin, temsirolimus (CC1-779), everolimus(RAD-001), ridaforolimus, AP23573 (Ariad), AZD8055 (AstraZeneca), BEZ235(Novartis), BGT226 (Novartis), XL765 (Sanofi Aventis), PF-4691502(Pfizer), GDC0980 (Genentech), SF1126 (Semafore) and OSI-027 (OSI)),oblimersen, gemcitabine, carminomycin, leucovorin, pemetrexed,cyclophosphamide, dacarbazine, procarbizine, prednisolone,dexamethasone, campathecin, plicamycin, asparaginase, aminopterin,methopterin, porfiromycin, melphalan, leurosidine, leurosine,chlorambucil, trabectedin, procarbazine, discodermolide, carminomycin,aminopterin, and hexamethyl melamine.

In certain embodiments, a compound provided herein, or apharmaceutically acceptable salt or isotopically labeled derivativethereof, or a pharmaceutical composition thereof, is used in combinationwith radiation therapy (RT). In certain embodiments, the compound isadministered in combination with surgery. In certain embodiments, thecompound is administered in combination with an immunotherapy.

For example, a compound of the present invention can be administered incombination with another therapeutic agent, such as anti-EGFR therapy,anti-HER2 therapy, anti-PD-1 therapy, anti-PD-L1 therapy, or irradiationtherapy.

In certain embodiments, a compound provided herein, or apharmaceutically acceptable salt or isotopically labeled derivativethereof, or a pharmaceutical composition thereof, is administered incombination with an anti-EGFR therapy (e.g., anti-EGFR monoclonalantibody (mAb), such as cetuximab). For example, provided herein is amethod of treating squamous cell carcinoma of the head and neck (SCCHN)in a subject comprising administering to said subject a compoundprovided herein, or a pharmaceutically acceptable salt or isotopicallylabeled derivative thereof, or a pharmaceutical composition thereof, incombination with an anti-EGFR (epidermal growth factor receptor) mAbtherapy. In certain embodiments, the anti-EGFR mAb is cetuximab (CTX).

In certain embodiments, a compound provided herein, or apharmaceutically acceptable salt or isotopically labeled derivativethereof, or a pharmaceutical composition thereof, is administered incombination with an anti-HER2 therapy (e.g., anti-HER2 monoclonalantibody (mAb), such as trastuzumab). For example, provided herein is amethod of treating breast cancer in a subject in need thereof comprisingadministering to said subject a compound provided herein, or apharmaceutically acceptable salt or isotopically labeled derivativethereof, or a composition thereof, in combination with an HER2 (humanepidermal growth factor receptor) mAb therapy. In certain embodiments,the anti-HER2 mAb is trastuzumab.

In certain embodiments, a compound provided herein, or apharmaceutically acceptable salt or isotopically labeled derivativethereof, or a pharmaceutical composition thereof, is administered incombination with an anti-PD-1 or anti-PD-L1 therapy (e.g., anti-PD-1 oranti-PD-L1 monoclonal antibody). For example, provided herein is amethod of treating colorectal cancer in a subject in need thereofcomprising administering to said subject a compound provided herein, ora pharmaceutically acceptable salt or isotopically labeled derivativethereof, or a composition thereof, in combination with an anti-PD-1 oranti-PD-L1 therapy (e.g., mAb therapy).

EXAMPLES Synthesis of Compounds General Procedures and Methods

The compound according to the present invention can be produced by themethods described in Examples below. However, these examples are onlyfor illustrative purposes, and the compound according to the presentinvention is not limited to the specific examples mentioned below in anyway.

In the Examples, unless specifically mentioned otherwise, the silica gelfor purification by using silica gel column chromatography was Hi-Flash™Column (Silica Gel, 30 μm 60 Å or 40 μm 60 Å, Yamazen Corporation), thesilica gel for purification by using NH silica gel column chromatographywas Chromatorex NH silica gel (Fuji Silysia Chemical LTD). Analyticalthin layer chromatography (TLC) was performed with TLC silica gel 60F₂₅₄, layer thickness 0.25 mm (Merck KGaA) or Chromatorex TLC NH silicagel F₂₅₄, layer thickness 0.25 mm (Fuji Silysia Chemical LTD). TLCplates were visualized by staining with p-anisaldehyde stain,phosphomolybdic acid stain or Hanessian's Stain.

All moisture sensitive reactions were conducted under an inertatmosphere. Reagents and solvents were commercial grade and were used assupplied, unless otherwise noted.

NMR spectra were recorded on a JEOL ECZ500R (500 MHz), JEOL ECZ400S (400MHz), Varian Inova 500 (500 MHz), Varian Mercury 400 (400 MHz) or BrukerAvance (600 MHz) spectrometer. Chemical shifts are reported in parts permillion (ppm). For ¹H NMR spectra (CDCl₃, C₆D₆, and/or CD₃OD), theresidual solvent peak was used as the internal reference (7.27 ppm inCDCl₃; 7.16 ppm in C₆D₆; 3.31 ppm in CD₃OD).

Analytical mass spectra (MS) results were obtained using a WatersAcquity UPLC equipped with a single quadrapole detector (SQ Detector 2)or LTQ Orbitrap XL™ (Thermoscientific).

High performance liquid chromatography (HPLC) was carried out withShimadzu LC-10AD on a UV spectrophotometric detector (200 nm, ShimadzuSPD-10A).

The abbreviations used herein are as follows: AIBN:2,2′-azobis(isobutyronitrile); Alloc: allyloxycarbonyl; 9-BBN:9-borabicyclo[3.3.1]nonane; Bu₃SnH: tri-normal-butyltin hydride;(+)—CSA: (1S)-(+)-10-Camphorsulfonic acid; DMAP:4-dimethylaminopyridine; DCM: dichloromethane; DDQ:2,3-dichloro-5,6-dicyano-1,4-benzoquinone; DIBAL: diisobutylaluminiumhydride; DMF: N,N-dimethylformamide; DMSO: dimethyl sulfoxide; Et₃N:triethylamine; EtOAc: ethyl acetate; HF-Pyridine: hydrogen fluoridepyridine; HPLC: high performance liquid chromatography; IPA: isopropylalcohol; MeCN: acetonitrile; MeOH: methanol; MPM: para-methoxybenzyl;PPh₃: triphenylphosphine; t-BuOH: tertiary-butyl alcohol; tBuLi:tertiary-butyl lithium; TBME: methyl tertiary-butyl ether; TBAF:tetrabutylammonium fluoride; TBS: tertiary-butyldimethylsilyl; THF:tetrahydrofuran; TMS: trimethylsilyl; Ts: para-toluenesulfonyl.

The synthetic intermediates disclosed herein are considered part of thepresent invention.

Example 1(4aR,5aS,6R,8aS,9aR)-2,2-di-tert-butyl-6-methyloctahydrofuro[2′,3′:5,6]pyrano[3,2-d][1,3,2]dioxasilin-7-ol

Under a nitrogen atmosphere, to the solution of Compound A-1:(4aR,5aS,6R,8aS,9aR)-2,2-di-tert-butyl-6-methylhexahydrofuro[2′,3′:5,6]pyrano[3,2-d][1,3,2]dioxasilin-7(8aH)-one(A-1 18.5 g, 54.0 mmol) obtained by the method written in OrganicLetters (2009), 11(2), 409-412 (CAS No; 1095280-04-8) in toluene (275mL) at −78° C., DIBAL (70.2 mL, 70.2 mmol, 1.0 M toluene solution) wasadded over 30 min. Then the reaction mixture was stirred at −78° C.After 90 min, the reaction was quenched with MeOH (4.37 mL) carefully at−78° C., then removed the cooling bath. Saturated potassium sodiumtartrate tetrahydrate solution (300 mL) was added to the reactionmixture, continued stirring for 2 hr at room temperature. The reactionmixture was poured into a separatory funnel, then the layers wereseparated. The aqueous layer was extracted with EtOAc (300 mL). Thecombined organic extracts were washed with brine (300 mL), dried overNa₂SO₄, filtered, concentrated under reduced pressure. The crude lactolwas used for the next reaction without purification.

Example 2(4aR,6S,7S,8aR)-6-((S)-but-3-en-2-yl)-2,2-di-tert-butylhexahydropyrano[3,2-d][1,3,2]dioxasilin-7-ol(Compound A-2)

Under a nitrogen atmosphere, to the suspension ofmethyltriphenylphosphonium bromide (73.30 g, 205.2 mmol) in THE (200mL), potassium tert-butoxide (17.27 g, 153.9 mmol) was added at −5° C.over 10 min, and then stirred for 60 min at −5° C. Solution of the crudelactol described in Example 1 in THE (40 mL) was transferred to thereaction mixture at −5° C. over 10 min, then stirred at −5° C. for 1 hr,at room temperature for 1 hr. The reaction mixture was quenched withice-water (400 mL), then diluted with TBME (400 mL) and then the layerswere separated. The aqueous layer was extracted with TBME (400 mL). Thecombined organic extracts were washed with brine (400 mL), dried overNa₂SO₄, filtered and concentrated under reduced pressure. The residuewas suspended with Heptane/EtOAc=1/1 (100 mL). The resulting suspensionwas filtered, rinsed with Heptane/EtOAc=1/1 (100 mL) to removetriphenylphosphine derived material. Then filtrate was concentratedunder reduced pressure. Flash chromatography of the residue on silicagel (400 g, Silica Gel 60, spherical, 40-50 μm, Kanto Chemical) using 0%to 20% EtOAc/Heptane gave the title compound (Compound A-2, 16.7 g, 90%yield).

¹H NMR (400 MHz, CHLOROFORM-d) δ ppm 1.03 (d, J=6.8 Hz, 3H) 1.05 (s, 9H)1.07 (s, 9H) 1.75 (dt, J=14.5, 3.0 Hz, 1H) 2.37 (dt, J=14.5, 2.9 Hz, 1H)2.65-2.76 (m, 1H) 3.03 (dd, J=9.8, 1.0 Hz, 1H) 3.31 (m, 1H) 3.69 (d,J=15.0 Hz, 1H) 3.75-3.79 (m, 1H) 4.16-4.31 (m, 2H) 4.41 (t, J=2.9 Hz,1H) 4.95-5.09 (m, 2H) 6.02 (ddd, J=17.3, 10.5, 6.3 Hz, 1H).

Example 3(4aR,6S,7S,8aR)-6-((S)-but-3-en-2-yl)-2,2-di-tert-butyl-7-((tert-butyldimethylsilyl)oxy)hexahydropyrano[3,2-d][1,3,2]dioxasiline(Compound A-3)

Under a nitrogen atmosphere, to a solution of Compound A-2:(4aR,6S,7S,8aR)-6-((S)-but-3-en-2-yl)-2,2-di-tert-butylhexahydropyrano[3,2-d][1,3,2]dioxasilin-7-ol(9.85 g, 28.8 mmol) described in Example 2 in DCM (150 mL) at 0° C. wereadded 2,6-lutidine (6.68 mL, 57.5 mmol) and tert-butyldimethylsilyltrifluoromethanesulfonate (9.25 mL, 40.3 mmol). The reaction mixture wasstirred at 0° C. for 30 min, then at room temperature for 2 hr. Thereaction mixture was diluted with diethyl ether. The organic layer waswashed with 0.5 N HCl aq, sat.NaHCO₃ aq and then brine. The combinedorganic layers were dried over MgSO₄, filtered (small amount of SiO₂)and concentrated under reduced pressure. Flash chromatography of theresidue on silica gel using 0% to 15% EtOAc/Heptane gave the titlecompound (Compound A-3, 12.0 g, 91% yield).

¹H NMR (500 MHz, CHLOROFORM-d) δ ppm 0.10 (s, 3H) 0.19 (s, 3H) 0.91 (s,9H) 0.96 (d, J=6.3 Hz, 3H) 1.02 (s, 9H) 1.06 (s, 9H) 1.73 (dt, J=15.0,4.0 Hz, 1H) 2.26 (dt, J=15.0, 2.5 Hz, 1H) 2.66-2.74 (m, 1H) 2.95 (dd,J=9.5, 2.2 Hz, 1H) 3.17 (m, 1H) 3.81-3.84 (m, 1H) 4.12-4.22 (m, 2H) 4.24(t, J=2.7 Hz, 1H) 4.93-5.06 (m, 2H) 6.08 (ddd, J=17.3, 10.5, 6.3 Hz,1H).

Example 4(2R,3R,5S,6S)-6-((S)-but-3-en-2-yl)-5-((tert-butyldimethylsilyl)oxy)-2-(hydroxymethyl)tetrahydro-2H-pyran-3-ol(Compound A-4)

Under a nitrogen atmosphere, to a solution of Compound A-3:(4aR,6S,7S,8aR)-6-((S)-but-3-en-2-yl)-2,2-di-tert-butyl-7-((tert-butyldimethylsilyl)oxy)hexahydropyrano[3,2-d][1,3,2]dioxasiline(12 g, 26.3 mmol) described in Example 3 in MeCN (120 mL) and DCM (40mL) at −10° C. was added pre-mixed solution of HF-Pyridine (4.0 mL) andpyridine (20 mL) in 20 mL of MeCN. The reaction mixture was stirred at−10° C. for 15 min, then at room temperature for 1 hr. The reactionmixture was quenched with sat.NaHCO₃ aq at 0° C. and diluted with DCM,then the layers were separated. The aqueous layer was extracted withDCM. The combined organic extracts were washed with brine. The combinedorganic layer was dried over MgSO₄, filtered and concentrated underreduced pressure. Flash chromatography of the residue on silica gelusing 15% to 60% EtOAc/Heptane gave the title compound (Compound A-4,8.4 g, Quant. yield).

¹H NMR (500 MHz, CHLOROFORM-d) δ ppm 0.13 (s, 3H) 0.19 (s, 3H) 0.94 (s,9H) 0.96 (d, J=6.8 Hz, 3H) 1.72 (dt, J=14.6, 2.9 Hz, 1H) 2.15 (dd,J=9.8, 2.4 Hz, 1H) 2.23 (dt, J=14.6, 2.9 Hz, 1H) 2.55-2.65 (m, 1H) 3.03(d, J=9.8 Hz, 1H) 3.41-3.46 (m, 1H) 3.49 (d, J=11.7 Hz, 1H) 3.62-3.72(m, 2H) 3.92 (ddd, J=11.7, 8.3, 2.4 Hz, 1H) 4.02 (t, J=2.7 Hz, 1H)5.01-5.12 (m, 2H) 5.93 (ddd, J=17.4, 10.4, 7.3 Hz, 1H).

Example 5(((2S,3S,5R,6R)-2-((S)-but-3-en-2-yl)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-3,5-diyl)bis(oxy))bis(tert-butyldimethylsilane)(Compound A-5)

Under a nitrogen atmosphere, to a solution of Compound A-4:(2R,3R,5S,6S)-6-((S)-but-3-en-2-yl)-5-((tert-butyldimethylsilyl)oxy)-2-(hydroxymethyl)tetrahydro-2H-pyran-3-ol(997 mg, 3.15 mmol) described in Example 4 in DCM (10 mL) at 5° C. wasadded 2,6-lutidine (1.83 mL, 15.8 mmol) and tert-butyldimethylsilyltrifluoromethanesulfonate (2.17 mL, 9.45 mmol). The reaction mixture wasstirred at room temperature for 5 hr. The reaction mixture was dilutedwith diethyl ether and quenched with sat. NaHCO₃ aq, then the layerswere separated. The combined organic extracts were successively washedwith 0.5 N HCl aq, sat.NaHCO₃ aq, and then brine. The organic layer wasdried over MgSO₄, filtered and concentrated under reduced pressure.Flash chromatography of the residue on silica gel using 0% to 5%EtOAc/Heptane (containing 1% Et₃N) gave the title compound (CompoundA-5, 1.69 g, 98% yield).

¹H NMR (500 MHz, CHLOROFORM-d) δ ppm 0.02-0.08 (m, 15H) 0.11 (s, 3H)0.89 (s, 9H) 0.90-0.92 (m, 18H) 0.94 (d, J=6.8 Hz, 3H) 1.82 (dt, J=14.9,4.8 Hz, 1H) 2.00 (dt, J=14.9, 2.9 Hz, 1H) 2.62-2.72 (m, 1H) 2.93 (dd,J=9.3, 2.0 Hz, 1H) 3.27-3.34 (m, 1H) 3.66-3.79 (m, 3H) 3.83-3.87 (m, 1H)4.91-5.07 (m, 2H) 6.11 (ddd, J=17.3, 10.7, 6.1 Hz, 1H).

Example 6(S)-3-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxymethyl)tetrahydro-2H-pyran-2-yl)butan-1-ol(Compound A-6)

To a solution of Compound A-5:(((2S,3S,5R,6R)-2-((S)-but-3-en-2-yl)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-3,5-diyl)bis(oxy))bis(tert-butyldimethylsilane)(1.32 g, 2.42 mmol) described in Example 5 in THE (10 mL) at 0° C. wasadded 9-BBN (9.69 mL, 0.5 M THE solution, 4.84 mmol). The reactionmixture was stirred at 0° C. for 1 hr and at room temperature for 1.5hr. 3.0 M NaOH aq (3 mL, 9.00 mmol) and hydrogen peroxide (35% in water,3 mL) were added to the reaction mixture at 0° C. The reaction mixturewas stirred at 0° C. for 30 min, then at room temperature for 1 hr. Thereaction mixture was quenched with sat. Na₂SO₃ aq and then the layerswere separated. The aqueous layer was extracted with EtOAc (3 times).The combined organic extracts were washed with brine, dried over Na₂SO₄,filtered and concentrated under reduced pressure. Flash chromatographyof the residue on silica gel using 0% to 20% EtOAc/Heptane gave thetitle compound (Compound A-6, 1.36 g, 100% yield).

¹H NMR (500 MHz, CHLOROFORM-d) δ ppm 0.03 (s, 3H) 0.05-0.08 (m, 12H)0.10 (s, 3H) 0.88 (d, J=6.8 Hz, 3H) 0.89-0.93 (m, 27H) 1.55-1.65 (m, 1H)1.82 (dt, J=15.4, 4.4 Hz, 1H) 1.87-1.96 (m, 1H) 1.97-2.03 (m, 1H)2.17-2.26 (m, 1H) 2.67 (dd, J=7.8, 3.9 Hz, 1H) 2.98-3.10 (m, 1H)3.34-3.40 (m, 1H) 3.59-3.86 (m, 6H) ESI-MS (m/z): 563.64 [M+H]⁺, 585.62[M+Na]⁺

Example 7(S)-3-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)butanal(Compound A-7)

Under a nitrogen atmosphere, to a solution of Compound A-6:(S)-3-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)butan-1-ol(1100 mg, 1.954 mmol) described in Example 6 in DCM (30 mL) at 5° C.were added NaHCO₃ (41.0 mg, 0.49 mmol) and Dess-Martin periodinane (1077mg, 2.54 mmol). The reaction mixture was stirred at room temperature.After 3 hr, the reaction mixture was diluted with DCM and quenched withsat.NaHCO₃ aq and sat. Na₂SO₃ aq, then the layers were separated. Theaqueous layer was extracted with DCM. The combined organic extracts werewashed with brine, dried over MgSO₄, filtered and concentrated underreduced pressure. Flash chromatography of the residue on silica gelusing 0% to 25% EtOAc/Heptane gave the title compound (Compound A-7, 950mg, 87% yield).

¹H NMR (500 MHz, CHLOROFORM-d) δ ppm 0.00 (s, 3H) 0.03-0.08 (m, 12H)0.11 (s, 3H) 0.88 (s, 9H) 0.91-0.92 (m, 21H) 1.82 (dt, J=15.0, 4.5 Hz,1H) 2.01 (dt, J=15.0, 2.5 Hz, 1H) 2.28 (ddd, J=16.0, 7.3, 2.4 Hz, 1H)2.53-2.58 (m, 1H) 2.74 (ddd, J=16.0, 5.5, 2.0 Hz, 1H) 2.94 (dd, J=9.0,1.7 Hz, 1H) 3.29 (td, J=5.9, 2.0 Hz, 1H) 3.68 (d, J=5.9 Hz, 2H)3.75-3.82 (m, 1H) 3.82-3.90 (m, 1H) 9.73 (t, J=2.4 Hz, 1H).

Example 8(2S,3S)-3-((4-methoxybenzyl)oxy)-2-methyl-5-(trimethylsilyl)pent-4-yn-1-yl4-methylbenzenesulfonate (Compound B-2)

Under a nitrogen atmosphere, to the solution of Compound B-1:(2S,3S)-3-((4-methoxybenzyl)oxy)-2-methyl-5-(trimethylsilyl)pent-4-yn-1-ol(11.08 g, 36.15 mmol) obtained by the method written in WO 9317690A1/U.S. Pat. No. 5,436,238 A (CAS No; 157323-41-6) in DCM (330 mL), Et₃N(12.6 mL, 90.4 mmol) and p-toluenesulfonyl chloride (8.27 g, 43.4 mmol)were added at room temperature. The reaction mixture was stirred at roomtemperature overnight. The mixture was washed with sat.NaHCO₃ and brine,dried over MgSO₄, filtered, then concentrated under reduced pressure.Flash chromatography of the residue on silica gel (Silica Gel 60,spherical, 40-50 μm, Kanto Chemical) using 0% to 10% EtOAc/Heptane gavethe title compound (Compound B-2, 17.7 g, 93% yield).

¹H NMR (500 MHz, CHLOROFORM-d) δ ppm 0.17 (s, 9H) 1.02 (d, J=6.8 Hz, 3H)2.10-2.18 (m, 1H) 2.44 (s, 3H) 3.82 (s, 3H) 3.99 (d, J=6.8 Hz, 1H)4.04-4.07 (m, 2H) 4.33 (d, J=11.2 Hz, 1H) 4.66 (d, J=11.2 Hz, 1H) 6.87(d, J=8.3 Hz, 2H) 7.21 (d, J=8.3 Hz, 2H) 7.33 (d, J=8.8 Hz, 2H) 7.77 (d,J=8.8 Hz, 2H).

Example 9((3S,4R)-5-iodo-3-((4-methoxybenzyl)oxy)-4-methylpent-1-yn-1-yl)trimethylsilane(Compound B-3)

Under a nitrogen atmosphere, to the solution of Compound B-2:(2S,3S)-3-((4-methoxybenzyl)oxy)-2-methyl-5-(trimethylsilyl)pent-4-yn-1-yl4-methylbenzenesulfonate (17.7 g, 38.4 mmol) described in Example 8 inDMF (360 mL), NaI (7.49 g, 50.0 mmol) was added at room temperature. Thereaction mixture was stirred at 80° C. for 2 hr. Another 2.0 g of NaIwas added to the reaction mixture. The reaction was stirred for 1.5 hrat 80° C., then cooled to room temperature. The mixture was diluted withdiethyl ether, washed with water and brine, dried over MgSO₄, filteredand concentrated under reduced pressure. Flash chromatography of theresidue on silica gel (Silica Gel 60, spherical, 40-50 μm, KantoChemical) using 10% to 20% EtOAc/Heptane gave the title compound(Compound B-3, 14.3 g, 89% yield).

¹H NMR (500 MHz, CHLOROFORM-d) δ ppm 0.21 (s, 9H) 1.10 (d, J=6.8 Hz, 3H)1.74-1.84 (m, 1H) 3.30-3.37 (m, 2H) 3.82 (s, 3H) 3.96 (d, J=7.3 Hz, 1H)4.44 (d, J=11.2 Hz, 1H) 4.73 (d, J=11.2 Hz, 1H) 6.89 (d, J=8.8 Hz, 2H)7.30 (d, J=8.8 Hz, 2H).

Example 10(2S,6S,7S)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-7-((4-methoxybenzyl)oxy)-6-methyl-9-(trimethylsilyl)non-8-yn-4-ol(Compound C-1)

Under an argon atmosphere, to a solution of Compound B-3:((3S,4R)-5-iodo-3-((4-methoxybenzyl)oxy)-4-methylpent-1-yn-1-yl)trimethylsilane(1408 mg, 3.382 mmol) described in Example 9 in diethyl ether (25 mL) at−78° C. was added tert-butyllithium (1.61M in pentane, 4.11 mL, 6.62mmol). The reaction mixture was stirred at −78° C. for 45 min. CompoundA-7:(S)-3-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)butanal(825 mg, 1.47 mmol) described in Example 7 in 5.0 mL of diethyl etherwas added to the reaction mixture at −78° C. The reaction mixture wasstirred at −78° C. for 60 min. The reaction mixture was quenched withsat.NH₄Cl aq. Organic layer was washed with brine, dried over Na₂SO₄,then concentrated under reduced pressure. Flash chromatography of theresidue on silica gel using 0% to 25% EtOAc/Heptane gave the titlecompound (Compound C-1, 1167 mg, 93% yield).

¹H NMR (500 MHz, CHLOROFORM-d) δ ppm 0.00-0.12 (m, 21H) 0.15-0.24 (m,6H) 0.82-0.96 (m, 30H) 1.03 (d, J=6.3 Hz, 3H) 1.38-1.55 (m, 1H)1.68-1.99 (m, 4H) 2.10-2.30 (m, 2H) 2.76-2.87 (m, 1H) 3.15 (d, J=9.75Hz, 1H) 3.33-3.38 (m, 1H) 3.56-4.02 (m, 9H) 4.37-4.50 (m, 1H) 4.64-4.78(m, 1H) 6.83-6.88 (m, 2H) 7.23-7.35 (m, 2H).

Example 11(2S,6S,7S,E)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-7-((4-methoxybenzyl)oxy)-6-methyl-9-(tributylstannyl)non-8-en-4-ol(Compound C-3)

To a solution of Compound C-1:(2S,6S,7S)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-7-((4-methoxybenzyl)oxy)-6-methyl-9-(trimethylsilyl)non-8-yn-4-ol(1165 mg, 1.37 mmol) described in Example 10 in MeOH (20 mL) at 20° C.was added K₂CO₃ (189 mg, 1.37 mmol). The reaction mixture was stirred at20° C. for 2 hr. The reaction mixture was diluted with EtOAc andquenched with sat.NH₄Cl aq, then the layers were separated. The aqueouslayer was extracted with EtOAc. The combined organic extracts werewashed with brine, dried over MgSO₄, filtered and concentrated underreduced pressure. Flash chromatography of the residue on silica gelusing 0% to 15% EtOAc/Heptane gave Compound C-2:(2S,6S,7S)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-7-((4-methoxybenzyl)oxy)-6-methylnon-8-yn-4-ol(1050 mg, 98% yield). ESI-MS (m/z): 801.50 [M+Na]⁺

Under a nitrogen atmosphere, to a solution of Compound C-2:(2S,6S,7S)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-7-((4-methoxybenzyl)oxy)-6-methylnon-8-yn-4-ol(780 mg, 1.00 mmol) obtained above in toluene (15 mL) at 20° C. wereadded tri-n-butyltin hydride (2.5 mL, 9.36 mmol) and2,2′-azobis(isobutyronitrile) (82 mg, 0.50 mmol). The reaction mixturewas stirred at 90° C. for 15 min. The reaction mixture was concentratedunder reduced pressure. Flash chromatography of the residue on silicagel using 0% to 15% EtOAc/Heptane gave the title compound (Compound C-3,970 mg, 91% yield).

¹H NMR (500 MHz, CHLOROFORM-d) δ ppm 0.02-0.13 (m, 18H) 0.84-0.96 (m,48H) 1.22-1.37 (m, 6H) 1.47-1.56 (m, 7H) 1.72-1.90 (m, 3H) 1.95-2.03 (m,1H) 2.11-2.28 (m, 2H) 2.82-2.86 (m, 1H) 3.08-3.15 (m, 1H) 3.33-3.40 (m,1H) 3.43-3.53 (m, 1H) 3.58-3.87 (m, 8H) 4.25-4.31 (m, 1H) 4.49-4.54 (m,1H) 5.83 (dd, J=19.3, 7.6 Hz, 1H) 6.05-6.13 (m, 1H) 6.83-6.90 (m, 2H)7.24 (d, J=8.8 Hz, 2H).

Example 12(2S,6S,7S,E)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methylnon-8-en-4-one(Compound C-4)

Under a nitrogen atmosphere, to a solution of Compound C-3:(2S,6S,7S)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-7-((4-methoxybenzyl)oxy)-6-methyl-9-(tributylstannyl)non-8-en-4-ol(970 mg, 0.91 mmol) described in Example 11 in 30 mL of DCM at 5° C. wasadded iodine (242 mg, 0.95 mmol) in DCM (6 mL) until it maintained theiodine color. The reaction mixture was quenched with sat.Na₂SO₃ aq andthe layers were separated. The aqueous layer was extracted with DCM. Thecombined organic extracts were washed with brine. The combined organiclayers were dried over Na₂SO₄, filtered and concentrated under reducedpressure. Flash chromatography of the residue on silica gel using 0% to25% EtOAc/Heptane gave(2S,6S,7S,E)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methylnon-8-en-4-ol(768 mg, 93% yield).

Under a nitrogen atmosphere, to a solution of(2S,6S,7S,E)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methylnon-8-en-4-ol(768 mg, 0.85 mmol) obtained above in DCM (25 mL) at room temperaturewas added NaHCO₃ (17.8 mg, 0.21 mmol) and Dess-Martin periodinane (485mg, 1.14 mmol). The reaction mixture was stirred at room temperature for4 hr. The reaction mixture was diluted with DCM and quenched withsat.NaHCO₃ aq and sat.Na₂SO₃ aq, and then the layers were separated. Theaqueous layer was extracted with DCM. The combined organic extracts werewashed with brine, dried over MgSO₄, filtered and concentrated underreduced pressure. Flash chromatography of the residue on silica gelusing 0% to 20% EtOAc/Heptane gave the title compound (Compound C-4, 776mg, Quant. yield).

¹H NMR (500 MHz, CHLOROFORM-d) δ ppm 0.00 (s, 3H) 0.03-0.07 (m, 12H)0.10 (s, 3H) 0.81 (d, J=6.3 Hz, 3H) 0.84 (d, J=6.3 Hz, 3H) 0.89 (s, 9H)0.91 (s, 9H) 0.92 (s, 9H) 1.80 (dt, J=15.0, 4.5 Hz, 1H) 1.99 (dt,J=15.0, 2.5 Hz, 1H) 2.17 (dd, J=16.6, 10.2 Hz, 1H) 2.20-2.29 (m, 2H)2.43-2.48 (m, 1H) 2.54 (d, J=12.7 Hz, 1H) 2.87 (dd, J=9.0, 1.7 Hz, 1H)2.99 (dd, J=16.6, 2.9 Hz, 1H) 3.27 (td, J=5.8, 2.4 Hz, 1H) 3.50-3.56 (m,1H) 3.66-3.74 (m, 2H) 3.75-3.78 (m, 1H) 3.80 (s, 3H) 3.81-3.85 (m, 1H)4.26 (d, J=11.7 Hz, 1H) 4.50 (d, J=11.7 Hz, 1H) 6.26 (d, J=14.6 Hz, 1H)6.42 (dd, J=14.6, 7.8 Hz, 1H) 6.87 (d, J=8.3 Hz, 2H) 7.21 (d, J=8.3 Hz,2H). ESI-MS (m/z): 927.39 [M+Na]⁺

Example 13(2S,6S,7S,E)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methylnon-8-en-4-one(Compound C-5)

To a solution of Compound C-4:(2S,6S,7S,E)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methylnon-8-en-4-one(600 mg, 0.66 mmol) described in Example 12 in THF (5.0 mL), IPA (5.0mL) and t-BuOH (5.0 mL) at 4° C. was added (1S)-(+)-10-Camphorsulfonicacid (154 mg, 0.66 mmol). The reaction mixture was stirred at 4° C. for20 hr. The reaction mixture was diluted with EtOAc and quenched withsat.NaHCO₃ aq, then the layers were separated. The aqueous layer wasextracted with EtOAc. The combined organic extracts were washed withbrine, dried over MgSO₄, filtered and concentrated under reducedpressure. Flash chromatography of the residue on silica gel using 0% to35% EtOAc/Heptane gave the title compound (Compound C-5, 500 mg, 95%yield).

¹H NMR (500 MHz, CHLOROFORM-d) δ ppm 0.01 (s, 3H) 0.04 (s, 3H) 0.07 (s,3H) 0.11 (s, 3H) 0.86-0.91 (m, 15H) 0.93 (s, 9H) 1.83 (dt, J=14.9, 4.8Hz, 1H) 1.93-2.00 (dt, J=14.9, 4.8 Hz, 1H) 2.19-2.26 (m, 1H) 2.29 (dd,J=14.9, 5.6 Hz, 1H) 2.39 (dd, J=16.6, 8.3 Hz, 1H) 2.44-2.66 (m, 4H) 2.91(dd, J=9.5, 1.7 Hz, 1H) 3.36-3.41 (m, 1H) 3.48 (td, J=11.3, 2.7 Hz, 1H)3.59 (t, J=7.1 Hz, 1H) 3.74-3.78 (m, 2H) 3.80 (s, 3H) 3.85 (m, 1H) 4.25(d, J=11.2 Hz, 1H) 4.46 (d, J=11.2 Hz, 1H) 6.28 (d, J=14.6 Hz, 1H) 6.43(dd, J=14.6, 7.8 Hz, 1H) 6.87 (d, J=8.8 Hz, 2H) 7.21 (d, J=8.8 Hz, 2H).ESI-MS (m/z): 813.30 [M+Na]⁺

Example 14((2R,3R,5S,6S)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-((2S,6S,7S,E)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methyl-4-oxonon-8-en-2-yl)tetrahydro-2H-pyran-2-yl)methyl4-methylbenzenesulfonate (Compound C-6)

Under a nitrogen atmosphere, to a solution of Compound C-5:(2S,6S,7S,E)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methylnon-8-en-4-one(500 mg, 0.63 mmol) described in Example 13 in DCM (10 mL) at 5° C. wereadded pyridine (2.54 mL, 31.6 mmol), p-toluenesulfonyl chloride (723 mg,3.79 mmol) and 4-dimethylaminopyridine (77 mg, 0.63 mmol). The reactionmixture was stirred at room temperature for 24 hr. p-Toluenesulfonylchloride (150 mg, 0.79 mmol) was added to the reaction mixture at roomtemperature. Then, the reaction mixture was stirred at room temperaturefor 8 hr. The reaction mixture was diluted with DCM and quenched withsat.NaHCO₃ aq, then the layers were separated. The aqueous layer wasextracted with DCM. The combined organic extracts were washed withbrine, dried over Na₂SO₄, filtered and concentrated under reducedpressure. Flash chromatography of the residue on silica gel using 0% to25% EtOAc/Heptane gave the title compound (Compound C-6, 560 mg, 94%yield).

¹H NMR (500 MHz, CHLOROFORM-d) δ ppm 0.01 (s, 3H) 0.04 (s, 3H) 0.04 (s,3H) 0.08 (s, 3H) 0.81 (d, J=6.8 Hz, 3H) 0.83 (s, 9H) 0.86 (d, J=6.8 Hz,3H) 0.89 (s, 9H) 1.81 (dt, J=14.9, 4.5 Hz, 1H) 1.91-1.96 (m, 1H)2.15-2.32 (m, 3H) 2.36-2.42 (m, 1H) 2.43 (s, 3H) 2.57 (d, J=12.7 Hz, 1H)2.77 (dd, J=16.6, 3.4 Hz, 1H) 2.87 (dd, J=9.0, 1.7 Hz, 1H) 3.53-3.58 (m,2H) 3.70-3.75 (m, 1H) 3.80-3.85 (m, 1H) 3.81 (s, 3H) 4.06 (dd, J=10.0,5.0 Hz, 1H) 4.08-4.16 (m, 1H) 4.28 (d, J=11.2 Hz, 1H) 4.51 (d, J=11.2Hz, 1H) 6.30 (d, J=14.6 Hz, 1H) 6.45 (dd, J=14.6, 7.8 Hz, 1H) 6.88 (d,J=8.8 Hz, 2H) 7.24 (d, J=8.8 Hz, 2H) 7.31 (d, J=8.3 Hz, 2H) 7.76 (d,J=8.3 Hz, 2H).

Example 15(2S,6S,7S,E)-2-((2S,3S,5R,6R)-6-(azidomethyl)-3,5-bis((tert-butyldimethylsilyl)oxy)tetrahydro-2H-pyran-2-yl)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methylnon-8-en-4-one(Compound C-7)

Under a nitrogen atmosphere, to a solution of Compound C-6:((2R,3R,5S,6S)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-((2S,6S,7S,E)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methyl-4-oxonon-8-en-2-yl)tetrahydro-2H-pyran-2-yl)methyl4-methylbenzenesulfonate (560 mg, 0.59 mmol) described in Example 14 inDMSO (5.6 mL) at 20° C. was added sodium azide (385 mg, 5.92 mmol). Thereaction mixture was stirred at 85° C. After 2 hr, sodium azide (100 mg,1.54 mmol) was added to the reaction mixture, then the reaction mixturewas stirred at 85° C. for 14 hr. The reaction mixture was diluted withEtOAc and quenched with H₂O, then the layers were separated. The organicextracts were successively washed with water and brine. The combinedorganic layers were dried over Na₂SO₄, filtered and concentrated underreduced pressure to give a crude residue. Flash chromatography of theresidue on silica gel using 0% to 15% EtOAc/Heptane gave the titlecompound (Compound C-7, 298 mg, 62% yield).

¹H NMR (500 MHz, CHLOROFORM-d) δ ppm 0.03 (s, 3H) 0.06 (s, 3H) 0.07 (s,3H) 0.10 (s, 3H) 0.84 (d, J=6.8 Hz, 3H) 0.85 (d, J=6.8 Hz, 3H) 0.91 (s,9H) 0.92 (s, 9H) 1.86 (dt, J=15.0, 4.7 Hz, 1H) 1.98 (dt, J=15.0, 2.9 Hz,1H) 2.19-2.32 (m, 3H) 2.41-2.49 (m, 1H) 2.58 (d, J=12.7 Hz, 1H) 2.94(dd, J=16.6, 2.9 Hz, 1H) 2.98 (dd, J=8.8, 2.0 Hz, 1H) 3.02 (dd, J=12.7,2.9 Hz, 1H) 3.47 (dt, J=8.8, 2.7 Hz, 1H) 3.49-3.54 (m, 1H) 3.63 (dd,J=12.7, 8.8 Hz, 1H) 3.69-3.73 (m, 1H) 3.81 (s, 3H) 3.83-3.88 (m, 1H)4.26 (d, J=11.7 Hz, 1H) 4.50 (d, J=11.7 Hz, 1H) 6.26 (d, J=14.6 Hz, 1H)6.42 (dd, J=14.6, 7.8 Hz, 1H) 6.87 (d, J=8.8 Hz, 2H) 7.22 (d, J=8.8 Hz,2H).

Example 16(((2R,3R,5S,6S)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-((2S,6S,7S,E)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methyl-4-oxonon-8-en-2-yl)tetrahydro-2H-pyran-2-yl)methyl)carbamate(Compound C-8)

To a solution of Compound C-7:(2S,6S,7S,E)-2-((2S,3S,5R,6R)-6-(azidomethyl)-3,5-bis((tert-butyldimethylsilyl)oxy)tetrahydro-2H-pyran-2-yl)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methylnon-8-en-4-one(298 mg, 0.37 mmol) described in Example 15 in THE (10 mL) and water(1.0 mL) at 20° C. was added triphenylphosphine (1437 mg, 5.478 mmol).The reaction mixture was stirred at 70° C. for 1.5 hr. The reactionmixture was concentrated under reduced pressure to give a crude amine.To a solution of the crude amine obtained above in THE (10 mL) at 5° C.were added Et₃N (0.51 mL, 3.66 mmol) and diallyl dicarbonate (341 mg,1.83 mmol). The reaction mixture was stirred at room temperature for 60min. The reaction mixture was concentrated under reduced pressure. Flashchromatography of the residue on silica gel using 0% to 25%EtOAc/Heptane gave the title compound (Compound C-8, 300 mg, 94% yield).

¹H NMR (500 MHz, CHLOROFORM-d) δ ppm 0.05-0.07 (m, 9H) 0.11 (s, 3H) 0.85(d, J=6.3 Hz, 3H) 0.87 (d, J=6.3 Hz, 3H) 0.90 (s, 9H) 0.93 (s, 9H) 1.80(dt, J=15.0, 4.4 Hz, 1H) 1.96 (dt, J=15.0, 2.8 Hz, 1H) 2.16-2.29 (m, 2H)2.32-2.39 (m, 1H) 2.53-2.60 (m, 3H) 2.86 (d, J=7.3 Hz, 1H) 3.04-3.11 (m,1H) 3.30-3.34 (m, 1H) 3.38-3.48 (m, 1H) 3.58 (t, J=7.1 Hz, 1H) 3.70-3.76(m, 1H) 3.80 (s, 3H) 3.81-3.84 (m, 1H) 4.25 (d, J=11.2 Hz, 1H) 4.46 (d,J=11.2 Hz, 1H) 4.53-4.63 (m, 2H) 5.19 (dd, J=10.7, 1.5 Hz, 1H) 5.32 (d,J=17.1 Hz, 1H) 5.47 (d, J=6.8 Hz, 1H) 5.88-5.99 (m, 1H) 6.28 (d, J=14.6Hz, 1H) 6.43 (dd, J=14.6, 7.8 Hz, 1H) 6.87 (d, J=8.8 Hz, 2H) 7.21 (d,J=8.8 Hz, 2H). ESI-MS (m/z): 896.34 [M+Na]⁺

Under a nitrogen atmosphere (in a glove box), to a solution of CompoundD-2:(S)—N-(2-(4-isopropyl-4,5-dihydrooxazol-2-yl)-6-methoxyphenyl)methanesulfonamide(155 mg, 0.497 mmol) obtained by the method written in Organic Letters(2002), 4 (25), 4431-4434 (CAS No; 546141-34-8) and1,8-bis(dimethylamino)naphthalene (107 mg, 0.497 mmol) in MeCN (0.75 mL)was added chromium(II) chloride (55.5 mg, 0.452 mmol) and then theresulting mixture was stirred in the glove box at room temperature for 1hr. The resulting green solution was added to a mixture of Compound C-8:allyl(((2R,3R,5S,6S)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-((2S,6S,7S,E)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methyl-4-oxonon-8-en-2-yl)tetrahydro-2H-pyran-2-yl)methyl)carbamate(99.0 mg, 0.113 mmol) described in Example 16, Compound D-1 (80.0 mg,0.09 mmol) obtained by the method written in Journal of the AmericanChemical Society (1992), 114 (8), 3162-3164 (CAS No; 157322-23-1),Compound D-3: dichloro(2,9-dimethyl-1,10-phenanthroline)nickel (0.46 mg,1.36 μmol) obtained by the method written in Journal of the AmericanChemical Society (2009), 131(42), 15387-15393 (CAS No; 21361-04-6) andlithium chloride (3.83 mg, 0.09 mmol). The reaction mixture was stirredin the glove box at room temperature for 60 min. The reaction mixturewas then taken out of the glove box, diluted with diethyl ether-EtOAc(5.0 mL-5.0 mL), then Florisil® (1600 mg, 15.94 mmol) (CAS No;1343-88-0) was added to the mixture. Then mixture was stirred at roomtemperature for 30 min. The mixture was filtered (Celite®), washed withEtOAc/Heptane=2/1, then filtrate was concentrated under reducedpressure. Flash chromatography of the residue on silica gel using 3% to55% EtOAc/Heptane gave the title compound (Compound D-4-1, 140 mg, 95%yield).

Example 18

Under a nitrogen atmosphere, to a solution of Compound D-4-1 (140 mg,0.09 mmol) described in Example 17 in DCM (5.0 mL) at 5° C. were addedNaHCO₃ (28.8 mg, 0.34 mmol) and Dess-Martin periodinane (72.7 mg, 0.17mmol). The reaction mixture was stirred at room temperature for 60 min.The reaction mixture was diluted with DCM and quenched with sat. NaHCO₃aq and sat. Na₂SO₃ aq, and then the layers were separated. The aqueouslayer was extracted with DCM. The combined organic extracts were washedwith brine, dried over Na₂SO₄, filtered and concentrated under reducedpressure. Flash chromatography of the residue on silica gel using 2% to60% EtOAc/Heptane gave the title compound (Compound D-5-1, 120 mg, 86%).

¹H NMR (500 MHz, BENZENE-d6) δ ppm 0.01-0.05 (m, 9H) 0.10-0.12 (m, 6H)0.15 (s, 3H) 0.76 (d, J=6.1 Hz, 3H) 0.96 (s, 9H) 1.02 (s, 9H) 1.04 (s,9H) 0.95-1.10 (m, 7H) 1.20 (d, J=7.3 Hz, 3H) 1.31-1.37 (m, 3H) 1.41 (dd,J=12.8, 4.9 Hz, 1H) 1.40-1.58 (m, 4H) 1.59-1.64 (m, 1H) 1.69-1.89 (m,3H) 1.90-1.99 (m, 2H) 2.02-2.25 (m, 8H) 2.26-2.48 (m, 6H) 2.49-2.70 (m,6H) 2.71-2.84 (m, 2H) 3.00-3.07 (m, 1H) 3.12-3.30 (m, 4H) 3.36 (s, 3H)3.40 (br.s, 1H) 3.44-3.53 (m, 2H) 3.65 (dd, J=6.4, 4.0 Hz, 1H) 3.69-3.84(m, 4H) 3.86-4.03 (m, 4H) 4.07-4.17 (m, 3H) 4.27-4.29 (m, 1H) 4.27 (d,J=11.0 Hz, 1H) 4.48-4.58 (m, 1H) 4.49 (d, J=11.0 Hz, 1H) 4.65-4.70 (m,2H) 4.68 (d, J=5.5 Hz, 1H) 4.74-4.86 (m, 2H) 4.78 (s, 1H) 4.93 (s, 1H)5.05 (d, J=10.4 Hz, 1H) 5.09 (br. s., 1H) 5.19 (br. s., 1H) 5.30 (dd,J=17.1, 1.2 Hz, 1H) 5.82 (d, J=8.0 Hz, 1H) 5.86-5.96 (m, 1H) 6.46 (d,J=15.9 Hz, 1H) 6.84-6.92 (m, 3H) 7.31 (d, J=8.6 Hz, 2H).

Example 19

Imidazole hydrochloride (155 mg, 1.48 mmol) was dissolved in DMF (2.9mL) to give a 0.5 M imidazole hydrochloride solution in DMF. 1.0 mL ofthis solution was mixed with 1.0 mL of TBAF (1.0 M, THF solution) togive a premixed solution of 0.5 M TBAF and 0.25 M imidazolehydrochloride in THF-DMF (1:1). Under a nitrogen atmosphere, to asolution of Compound D-5-1 (80.0 mg, 0.05 mmol) described in Example 18in DMF (7.0 mL) at 20° C. were added 0.588 mL of premixed solution ofTBAF (0.5 M) and imidazole hydrochloride (0.25 M) in THF-DMF (1:1)prepared above. The reaction mixture was stirred at room temperature for14 hr. 1.6 g of CaCO₃ and 4.0 g of Dowex® 50WX8 (hydrogen form, 200-400mesh, SIGMA-ALDRICH) were added to the reaction mixture. The mixture wasstirred at room temperature for 2 hr. Then mixture was diluted withEtOAc, then filtered (Celite®), washed with EtOAc. Filtrate wasconcentrated under reduced pressure to give a crude residue. 1000 mg ofCaCO₃ and 2.25 g of Dowex® 50WX8 were added to the EtOAc (6.0 mL)solution of the crude residue. The mixture was stirred at roomtemperature for 2.5 hr. Then mixture was diluted with EtOAc, filtered(Celite®), washed with EtOAc. Filtrate was concentrated under reducedpressure to give a crude residue (63.0 mg). To a solution of the cruderesidue (63.0 mg) obtained above in DCM (6.0 mL), t-BuOH (0.6 mL) and pH7 Phosphate Buffer (0.6 mL, 1/15 M) at room temperature was added DDQ(111 mg, 0.49 mmol). The reaction mixture was stirred at roomtemperature for 45 min. The reaction mixture was quenched with sat.NaHCO₃ aq, then diluted with DCM and the layers were separated. Theaqueous layer was extracted with DCM (3 times). The combined organicextracts were dried over Na₂SO₄, filtered and concentrated under reducedpressure. Flash chromatography of the residue on NH silica gel using 10%to 100% EtOAc/Heptane, then 10% MeOH/EtOAc gave a roughly purified titlecompound (Compound D-6-1, 15.0 mg, 27%).

¹H NMR (500 MHz, METHANOL-d4) δ ppm 0.97 (d, J=7.0 Hz, 3H) 0.97 (d,J=7.0 Hz, 3H) 1.00-1.02 (m, 1H) 1.05 (d, J=7.3 Hz, 3H) 1.09 (d, J=6.3Hz, 3H) 1.31-1.45 (m, 6H) 1.46-1.63 (m, 5H) 1.64-1.75 (m, 3H) 1.80-1.86(m, 2H) 1.87-1.93 (m, 2H) 1.94-2.11 (m, 9H) 2.13-2.27 (m, 8H) 2.33 (d,J=2.4 Hz, 2H) 2.39 (dd, J=13.4, 6.1 Hz, 1H) 2.44 (dd, J=17.6, 2.0 Hz,1H) 2.55 (dd, J=17.6, 9.3 Hz, 1H) 2.75-2.84 (m, 1H) 2.97 (dd, J=9.3, 2.0Hz, 1H) 3.21 (dd, J=6.6, 4.6 Hz, 1H) 3.32 (m, 1H) 3.41-3.46 (m, 1H) 3.57(br. s., 1H) 3.60 (d, J=11.7 Hz, 1H) 3.67-3.74 (m, 2H) 3.78 (br. s., 1H)3.86-3.90 (m, 2H) 3.97 (d, J=2.4 Hz, 1H) 4.02-4.11 (m, 4H) 4.17 (dd,J=6.6, 4.6 Hz, 1H) 4.23 (dd, J=11.5, 2.2 Hz, 1H) 4.29 (br.s, 1H) 4.31(td, J=9.3, 3.9 Hz, 1H) 4.44 (d, J=10.2 Hz, 1H) 4.51 (d, J=5.4 Hz, 2H)4.59 (t, J=4.9 Hz, 1H) 4.61 (dd, J=7.3, 4.9 Hz, 1H) 4.69 (t, J=4.6 Hz,1H) 4.80 (s, 1H) 4.85-4.87 (m, 1H) 5.01 (s, 1H) 5.05 (s, 1H) 5.16 (dd,J=10.7, 1.0 Hz, 1H) 5.28 (dd, J=17.1, 2.0 Hz, 1H) 5.92 (m, 1H). ESI-MS(m/z): 1172.57 [M+Na]⁺

Example 20

Under a nitrogen atmosphere, to a solution of Compound D-6-1 (15.0 mg,0.013 mmol) described in Example 19, pyrrolidine (10.8 μL, 0.13 mmol) inDCM (2.0 mL) at room temperature was addedtetrakis(triphenylphosphine)palladium(0) (7.53 mg, 6.52 μmol). Thereaction mixture was stirred at room temperature for 30 min. Thereaction mixture was concentrated under reduced pressure. Flashchromatography of the residue on NH silica gel using 50% EtOAc/Heptane,then 0% to 20% MeOH/EtOAc to give a roughly purified product. Obtainedroughly purified product was purified by HPLC to give the title compound(D-7-1, 7.0 mg, 47%, retention time=13.8 min).

HPLC Conditions:

Column: YMC Pak Pro C18 (20 mm×250 mm)

Detection wavelength: 200 nm

Column temperature: room temperature

Mobile phase: MeCN-Water (0.05% AcOH)

Flow rate: 8 mL/min

Eluate:

MeCN/Water 25% (iso, 2 min), then

MeCN/Water 25% to 60% (Gradient, 20 min)

¹H NMR (500 MHz, METHANOL-d4) δ ppm 0.99 (d, J=6.7 Hz, 3H) 1.00-1.03 (m,1H) 1.04 (d, J=7.3 Hz, 3H) 1.06 (d, J=7.3 Hz, 3H) 1.10 (d, J=6.1 Hz, 3H)1.29-1.63 (m, 10H) 1.65-1.78 (m, 3H) 1.79-1.89 (m, 2H) 1.92-2.12 (m,10H) 1.93 (s, 3H) 2.13-2.36 (m, 9H) 2.41 (dd, J=13.5, 6.1 Hz, 1H) 2.45(dd, J=17.6, 2.2 Hz, 1H) 2.56 (dd, J=17.6, 9.8 Hz, 1H) 2.75-2.84 (m, 1H)2.98 (dd, J=9.8, 1.8 Hz, 1H) 3.12 (dd, J=12.8, 3.7 Hz, 1H) 3.22 (dd,J=6.4, 4.6 Hz, 1H) 3.26 (dd, J=13.2, 7.8 Hz, 1H) 3.39 (d, J=1.8 Hz, 1H)3.61 (d, J=12.8 Hz, 1H) 3.63-3.68 (m, 2H) 3.68-3.76 (m, 2H) 3.81-3.94(m, 3H) 4.00 (d, J=2.5 Hz, 1H) 4.03-4.15 (m, 4H) 4.18 (dd, J=6.4, 4.6Hz, 1H) 4.25 (ddd, J=11.0, 4.3, 1.8 Hz, 1H) 4.27-4.36 (m, 2H) 4.46 (d,J=11.0 Hz, 1H) 4.57-4.65 (m, 2H) 4.70 (t, J=4.6 Hz, 1H) 4.81 (d, J=1.2Hz, 1H) 5.02 (br. s, 1H) 5.06 (d, J=1.8 Hz, 1H). ESI-MS (m/z): 1066.96[M+H]⁺, 1090.19 [M+Na]⁺

Compound (1) (salt free form of Compound D-7-1): ¹H NMR (600 MHz,METHANOL-d4) δ ppm 0.98 (d, J=7.2 Hz, 3H) 1.00 (d, J=6.8 Hz, 3H) 1.02(m, 1H) 1.05 (d, J=6.8 Hz, 3H) 1.09 (d, J=6.4 Hz, 3H) 1.28-1.45 (m, 5H)1.46-1.59 (m, 4H) 1.57-1.63 (m, 1H) 1.65-1.71 (m, 1H) 1.70-1.75 (m, 2H)1.79-1.86 (m, 2H) 1.91 (dt, J=14.9, 3.1 Hz, 1H) 1.94-2.11 (m, 8H)2.14-2.34 (m, 9H) 2.39 (dd, J=13.2, 6.0 Hz, 1H) 2.44 (dd, J=17.4, 1.9Hz, 1H) 2.56 (dd, J=17.6, 9.6 Hz, 1H) 2.69 (dd, J=13.2, 4.2 Hz, 1H) 2.79(ddq, J=15.9, 7.6, 2.0 Hz, 1H) 2.92 (dd, J=13.2, 8.3 Hz, 1H) 2.97 (dd,J=9.6, 1.7 Hz, 1H) 3.21 (dd, J=6.4, 4.9 Hz, 1H) 3.29 (m, 1H) 3.34 (dd,J=8.3, 4.15 Hz, 1H) 3.58 (br. s., 1H) 3.60 (br.d, J=11.3 Hz, 1H)3.68-3.73 (m, 2H) 3.80 (br. s., 1H) 3.84-3.90 (m, 2H) 3.98 (d, J=2.3 Hz,1H) 4.03-4.13 (m, 4H) 4.17 (dd, J=6.4, 4.9 Hz, 1H) 4.24 (ddd, J=11.3,4.5, 1.5 Hz, 1H) 4.29 (dd, J=4.0, 1.9 Hz, 1H) 4.32 (td, J=10.2, 4.2 Hz,1H) 4.44 (br. d, J=11.0 Hz, 1H) 4.59 (t, J=4.5 Hz, 1H) 4.62 (dd, J=7.4,4.7 Hz, 1H) 4.69 (t, J=4.7 Hz, 1H) 4.80 (br. s., 1H) 4.87 (s, 1H) 5.00(br. s., 1H) 5.05 (br.d, J=1.1 Hz, 1H)

ESI-MS (m/z): 1066.57 [M+H]⁺, 1088.55 [M+Na]⁺

Synthesis of Compound (D-6)

Example 21(S)-3-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)butanal(Compound A-7)

The title compound A-7 was obtained from compound A-1:(4aR,5aS,6R,8aS,9aR)-2,2-di-tert-butyl-6-methylhexahydrofuro[2′,3′:5,6]pyrano[3,2-d][1,3,2]dioxasilin-7(8aH)-oneprepared by the method written in Organic Letters (2009), 11(2), 409-412(CAS No; 1095280-04-8) as shown in Scheme A.

¹H NMR (500 MHz, CHLOROFORM-d) ppm 0.00 (s, 3H) 0.03-0.08 (m, 12H) 0.11(s, 3H) 0.88 (s, 9H) 0.91-0.92 (m, 21H) 1.82 (dt, J=15.0, 4.5 Hz, 1H)2.01 (dt, J=15.0, 2.5 Hz, 1H) 2.28 (ddd, J=16.0, 7.3, 2.4 Hz, 1H)2.53-2.58 (m, 1H) 2.74 (ddd, J=16.0, 5.5, 2.0 Hz, 1H) 2.94 (dd, J=9.0,1.7 Hz, 1H) 3.29 (td, J=5.9, 2.0 Hz, 1H) 3.68 (d, J=5.9 Hz, 2H)3.75-3.82 (m, 1H) 3.82-3.90 (m, 1H) 9.73 (t, J=2.4 Hz, 1H)

Example 22((3S,4R)-5-iodo-3-((4-methoxybenzyl)oxy)-4-methylpent-1-yn-1-yl)trimethylsilane(Compound B-3)

The title compound B-3 was obtained from compound B-1:(2S,3S)-3-((4-methoxybenzyl)oxy)-2-methyl-5-(trimethylsilyl)pent-4-yn-1-olprepared by the method written in WO 93/17690 A1/U.S. Pat. No. 5,436,238A (CAS No; 157323-41-6) as shown in Scheme B.

¹H NMR (500 MHz, CHLOROFORM-d) δ ppm 0.21 (s, 9H) 1.10 (d, J=6.8 Hz, 3H)1.74-1.84 (m, 1H) 3.30-3.37 (m, 2H) 3.82 (s, 3H) 3.96 (d, J=7.3 Hz, 1H)4.44 (d, J=11.2 Hz, 1H) 4.73 (d, J=11.2 Hz, 1H) 6.89 (d, J=8.8 Hz, 2H)7.30 (d, J=8.8 Hz, 2H).

Example 23(2S,6S,7S,E)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methylnon-8-en-4-one(Compound C-4)

The title compound C-4 was obtained from Compound A-7:(S)-3-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)butanaland Compound B-3:((3S,4R)-5-iodo-3-((4-methoxybenzyl)oxy)-4-methylpent-1-yn-1-yl)trimethylsilaneaccording to the procedure for the preparation of Compound C-8, as shownin Scheme C.

¹H NMR (500 MHz, CHLOROFORM-d) δ ppm 0.00 (s, 3H) 0.03-0.07 (m, 12H)0.10 (s, 3H) 0.81 (d, J=6.3 Hz, 3H) 0.84 (d, J=6.3 Hz, 3H) 0.89 (s, 9H)0.91 (s, 9H) 0.92 (s, 9H) 1.80 (dt, J=15.0, 4.5 Hz, 1H) 1.99 (dt,J=15.0, 2.5 Hz, 1H) 2.17 (dd, J=16.6, 10.2 Hz, 1H) 2.20-2.29 (m, 2H)2.43-2.48 (m, 1H) 2.54 (d, J=12.7 Hz, 1H) 2.87 (dd, J=9.0, 1.7 Hz, 1H)2.99 (dd, J=16.6, 2.9 Hz, 1H) 3.27 (td, J=5.8, 2.4 Hz, 1H) 3.50-3.56 (m,1H) 3.66-3.74 (m, 2H) 3.75-3.78 (m, 1H) 3.80 (s, 3H) 3.81-3.85 (m, 1H)4.26 (d, J=11.7 Hz, 1H) 4.50 (d, J=11.7 Hz, 1H) 6.26 (d, J=14.6 Hz, 1H)6.42 (dd, J=14.6, 7.8 Hz, 1H) 6.87 (d, J=8.3 Hz, 2H) 7.21 (d, J=8.3 Hz,2H). ESI-MS (m/z): 927.4 [M+Na]⁺

Example 24

Under a nitrogen atmosphere (in a glove box), to a solution of CompoundD-2:(S)—N-(2-(4-isopropyl-4,5-dihydrooxazol-2-yl)-6-methoxyphenyl)methanesulfonamide(29.1 mg, 0.093 mmol) obtained by the method written in Organic Letters(2002), 4 (25), 4431-4434 (CAS No; 546141-34-8) and1,8-bis(dimethylamino)naphthalene (20.0 mg, 0.093 mmol) in MeCN (0.3 mL)was added chromium(II) chloride (10.4 mg, 0.085 mmol) and then theresulting mixture was stirred in the glove box at room temperature for 1hr. The resulting solution was added to the pre-mixture of Compound C-4:(2S,6S,7S,E)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methylnon-8-en-4-one(18.4 mg, 0.02 mmol) described in Example 23, Compound D-1 (15.0 mg,0.017 mmol) obtained by the method written in Journal of the AmericanChemical Society (1992), 114 (8), 3162-3164 (CAS No; 157322-23-1),Compound D-3: dichloro(2,9-dimethyl-1,10-phenanthroline)nickel (0.09 mg,0.254 μmol) obtained by the method written in Journal of the AmericanChemical Society (2009), 131(42), 15387-15393 (CAS No; 21361-04-6) andlithium chloride (1.0 mg, 0.024 mmol). The reaction mixture was stirredin the glove box at room temperature for 60 min. The reaction mixturewas then taken out from the glove box, diluted with diethyl ether (0.45mL), then Florisil® (300 mg, 2.99 mmol) (CAS No; 1343-88-0) was added tothe mixture. Then mixture was stirred at room temperature for 30 min.The mixture was filtered (Celite®), washed with EtOAc/Heptane=1/1, thenfiltrate was concentrated under reduced pressure. Flash chromatographyof the residue on silica gel using 3% to 45% EtOAc/Heptane gave thetitle compound (Compound D-4, 25.5 mg, 90% yield).

Example 25

Under a nitrogen atmosphere, to a solution of Compound D-4 (25.0 mg,0.015 mmol) described in Example 24 in DCM (1.5 mL) at 0° C. were addedNaHCO₃ (0.38 mg, 4.51 μmol) and Dess-Martin periodinane (12.7 mg, 0.03mmol). The reaction mixture was stirred at room temperature for 5 hr.Dess-Martin periodinane (4.0 mg, 9.43 μmol) was added to the reactionmixture, then the reaction mixture was stirred at room temperature for 3hr.

The reaction mixture was diluted with DCM and quenched with sat.NaHCO₃aq and sat.Na₂SO₃ aq, and then the layers were separated. The aqueouslayer was extracted with DCM. The combined organic extracts were washedwith brine, dried over Na₂SO₄, filtered and concentrated under reducedpressure. Flash chromatography of the residue on silica gel using 3% to40% EtOAc/Heptane gave the title compound (Compound D-5, 20 mg, 80%). ¹HNMR (500 MHz, CHLOROFORM-d) δ ppm −0.01-0.01 (m, 6H) 0.03-0.08 (m, 15H)0.10 (s, 3H) 0.80 (d, J=6.3 Hz, 3H) 0.84 (d, J=6.8 Hz, 3H) 0.87 (s, 9H)0.88 (s, 9H) 0.91 (s, 9H) 0.91 (s, 9H) 0.94-0.98 (m, 1H) 1.04 (d, J=6.8Hz, 3H) 1.07 (d, J=6.3 Hz, 3H) 1.29-1.51 (m, 5H) 1.60-1.68 (m, 3H)1.68-1.77 (m, 2H) 1.80 (dt, J=14.8, 4.6 Hz, 1H) 1.86-2.03 (m, 7H)2.06-2.27 (m, 9H) 2.29 (d, J=2.4 Hz, 2H) 2.37 (dd, J=16.3, 4.6 Hz, 1H)2.41-2.50 (m, 1H) 2.54 (d, J=13.7 Hz, 1H) 2.64 (dd, J=16.6, 7.3 Hz, 1H)2.76-2.90 (m, 3H) 2.91-3.04 (m, 3H) 3.24-3.30 (m, 1H) 3.41 (dd, J=5.6,4.1 Hz, 1H) 3.54-3.59 (m, 1H) 3.66-3.75 (m, 3H) 3.75-3.78 (m, 1H) 3.80(s, 3H) 3.81-3.84 (m, 1H) 3.86-3.96 (m, 3H) 4.03-4.09 (m, 2H) 4.20 (dd,J=6.8, 4.9 Hz, 1H) 4.24 (d, J=11.2 Hz, 1H) 4.33 (br. s., 2H) 4.39-4.45(m, 2H) 4.47 (d, J=11.7 Hz, 1H) 4.57 (d, J=7.3 Hz, 1H) 4.60 (t, J=4.4Hz, 1H) 4.69 (t, J=4.6 Hz, 1H) 4.75 (s, 1H) 4.83 (s, 1H) 5.00 (d, J=13.2Hz, 2H) 6.23 (d, J=16.1 Hz, 1H) 6.66 (dd, J=16.1, 6.8 Hz, 1H) 6.87 (d,J=8.8 Hz, 2H) 7.21 (d, J=8.8 Hz, 2H).

Example 26

One milliliter of TBAF (1.0 mmol, 1.0 M THE solution) was co-evaporatedwith toluene (3×1 mL). To the residue was added imidazole hydrochloride(52 mg, 0.5 mmol) and DMF (2.0 mL) to give a premixed solution of 0.5 MTBAF and 0.25 M imidazole hydrochloride in DMF. Under a nitrogenatmosphere, to a solution of Compound D-5 (20.7 mg, 0.012 mmol)described in Example 25 in DMF (0.9 mL) and MeOAc (0.1 mL) at roomtemperature was added 0.241 mL of premixed solution of TBAF (0.5 M) andimidazole hydrochloride (0.25 M) in DMF prepared above. The reactionmixture was stirred at room temperature for 18 hr. The reaction mixturewas added with 210 mg of CaCO₃ and 520 mg of Dowex® 50WX8 (hydrogenform, 200-400 mesh, SIGMA-ALDRICH). The mixture was diluted with EtOAc(0.3 mL) and stirred at room temperature for 1 hr. The mixture wasdiluted with EtOAc (3 mL), then filtered through Celite®, washed withEtOAc. The filtrate was concentrated under reduced pressure to give acrude residue. The residue was added EtOAc (3 mL). The solution wasadded with 210 mg of CaCO₃ and 520 mg of Dowex® 50WX8. The mixture wasstirred at room temperature for 1 hr. The mixture was diluted withEtOAc, filtered through Celite®, washed with EtOAc. The filtrate wasconcentrated under reduced pressure to give a crude residue (16.7 mg).To a solution of the crude residue (16.7 mg) obtained above in DCM (2.1mL), t-BuOH (0.21 mL), and pH7 Phosphate Buffer (0.21 mL, 1/15 M) atroom temperature was added DDQ (28.3 mg, 0.125 mmol). The reactionmixture was stirred vigorously at room temperature for 50 min. Thereaction mixture was poured into sat. NaHCO₃ aq. (8 mL) with vigorousstirring at room temperature, then diluted with DCM (10 mL) and thelayers were separated. The aqueous layer was extracted with DCM (2×5mL). The combined organic extracts were dried over Na₂SO₄, filtered andconcentrated under reduced pressure. Flash chromatography of the residueon NH silica gel (FUJI SILYSIA CHEMICAL LTD, Chromatorex® NH silica gel)using 0% to 100% EtOAc/Heptane, then 5% MeOH/EtOAc gave a roughlypurified title compound (4.9 mg). The obtained product was purified byHPLC to give the title compound (Compound D-6, 2.2 mg, 16.5%). Retentiontime=8.7 min

HPLC Conditions

Column: YMC Pak Pro C18 (10 mm I.D.×250 mm)

Detection wavelength: 205 nm

Column temperature: room temperature

Mobile phase: MeCN-Water

Flow rate: 4.7 mL/min

Eluate:

-   -   MeCN/Water 60% (iso, 2 min), then    -   MeCN/Water 60% to 99% (gradient, 10 min), then    -   MeCN/Water 99% (iso, 11 min)

¹H NMR (500 MHz, METHANOL-d4) δ ppm 0.97 (d, J=6.8 Hz, 3H) 0.99 (d,J=7.3 Hz, 3H) 1.00-1.05 (m, 1H) 1.05 (d, J=7.3 Hz, 3H) 1.09 (d, J=6.8Hz, 3H) 1.30-1.57 (m, 8H) 1.57-1.62 (m, 1H) 1.64-1.70 (m, 1H) 1.70-1.75(m, 2H) 1.78-1.86 (m, 2H) 1.90 (ddd, J=14.8, 3.2, 3.2 Hz, 1H) 1.94-2.12(m, 9H) 2.13-2.25 (m, 4H) 2.25-2.34 (m, 5H) 2.39 (dd, J=13.2, 5.8 Hz,1H) 2.44 (dd, J=17.4, 2.3 Hz, 1H) 2.55 (dd, J=17.6, 9.7 Hz, 1H)2.76-2.83 (m, 1H) 2.97 (dd, J=9.7, 1.9 Hz, 1H) 3.21 (dd, J=6.6, 4.6 Hz,1H) 3.30 (m, 1H) 3.41 (dd, J=6.1, 6.1 Hz, 1H) 3.60 (d, J=11.7 Hz, 1H)3.65 (d, J=2.9 Hz, 1H) 3.65-3.68 (m, 2H) 3.68-3.74 (m, 2H) 3.80 (br. s.,1H) 3.84-3.91 (m, 2H) 3.98 (dd, J=4.7, 2.2 Hz, 1H) 4.03-4.13 (m, 4H)4.17 (dd, J=6.6, 4.6 Hz, 1H) 4.23 (ddd, J=11.6, 4.6, 2.0 Hz, 1H)4.28-4.30 (m, 1H) 4.32 (dd, J=10.2, 4.2 Hz, 1H) 4.44 (d, J=11.2 Hz, 1H)4.59 (dd, J=4.4, 4.4 Hz, 1H) 4.61 (dd, J=7.6, 4.8 Hz, 1H) 4.69 (dd,J=4.6, 4.6 Hz, 1H) 4.80 (d, J=1.2 Hz, 1H) 4.86-4.87 (m, 1H) 5.01 (br.s., 1H) 5.06 (d, J=1.5 Hz, 1H). ESI-MS (m/z): 1089.5 [M+Na]⁺

Synthesis of Compound (E-2) Example 27

The title compound E-2 was obtained from compound E-1:(2S,6S,7S,E)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methylnon-8-en-4-ol(CAS No; 157323-29-0) prepared by the method written in WO 93/17690 A1,and Compound D-1 (CAS No; 157322-23-1) prepared by the method written inJournal of the American Chemical Society 1992, 114, 3162-3164, using thesame procedures described for the preparation of compounds D-4, D-5, andD-6 (Example 24, 25, and 26). Retention time=14.7 min.

HPLC Conditions

Column: YMC Pak Pro C18 (4.6 mm I.D.×250 mm)

Detection wavelength: 200 nm

Column temperature: room temperature

Mobile phase: MeCN-Water

Flow rate: 1 ml/min

Eluate:

-   -   MeCN/Water 40% to 98% (Gradient, 20 min), then    -   MeCN/Water 98% (iso, 10 min)

¹H NMR (600 MHz, METHANOL-d4) δ ppm 0.96-0.98 (m, 6H) 1.01 (d, J=11.7Hz, 1H) 1.05 (d, J=6.8 Hz, 3H) 1.09 (d, J=6.4 Hz, 3H) 1.30-1.56 (m, 7H)1.63-1.71 (m, 3H) 1.72 (d, J=10.6 Hz, 1H) 1.79-1.86 (m, 2H) 1.89-2.12(m, 11H) 2.13-2.21 (m, 3H) 2.22-2.32 (m, 6H) 2.39 (dd, J=13.2, 6.0 Hz,1H) 2.44 (dd, J=17.8, 2.3 Hz, 1H) 2.55 (dd, J=17.4, 9.4 Hz, 1H) 2.79(dd, J=15.7, 5.5 Hz, 1H) 2.97 (dd, J=9.4, 1.5 Hz, 1H) 3.21 (dd, J=6.4,4.9 Hz, 1H) 3.28 (br. s., 2H) 3.47 (br. s., 1H) 3.51 (dd, J=9.1, 3.8 Hz,1H) 3.60 (d, J=11.7 Hz, 1H) 3.66-3.70 (m, 1H) 3.67-3.71 (m, 1H)3.68-3.73 (m, 2H) 3.78 (br. s., 1H) 3.86 (br. s., 1H) 3.86-3.90 (m, 1H)3.98 (q, J=2.3 Hz, 1H) 4.03-4.08 (m, 1H) 4.07-4.10 (m, 1H) 4.09-4.12 (m,1H) 4.10-4.12 (m, 2H) 4.17 (dd, J=6.4, 4.9 Hz, 1H) 4.23 (ddq, J=11.0,4.2, 1.9 Hz, 1H) 4.29 (t, J=1.9 Hz, 1H) 4.32 (td, J=10.6, 4.5 Hz, 1H)4.44 (d, J=11.0 Hz, 1H) 4.59 (t, J=4.3 Hz, 1H) 4.62 (dd, J=7.4, 4.7 Hz,1H) 4.69 (t, J=4.7 Hz, 1H) 4.84 (br. s., 1H) 4.87 (br. s., 1H) 5.00 (br.s., 1H) 5.05 (br. s., 1H). ESI-MS (m/z): 1103.6 [M+Na]⁺

Synthesis of Compound (G-4)

Example 28

The title compound G-4 was obtained as an acetic acid salt from compoundF-1:allyl-(2-((2R,3R,5S,6S)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-((2S,6S,7S,E)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methyl-4-oxonon-8-en-2-yl)tetrahydro-2H-pyran-2-yl)ethyl)carbamate(prepared by the procedure described in Scheme F), and compound D-1 (CASNo; 157322-23-1) prepared by the method written in Journal of theAmerican Chemical Society 1992, 114, 3162-3164, the entire contents ofwhich is incorporated herein by reference, based on the proceduredescribed in Scheme G. Retention time=10.7 min.

HPLC Conditions

Column: YMC Pak Pro C18 (10 mm I.D.×250 mm)

Detection wavelength: 200 nm

Column temperature: room temperature

Mobile phase: MeCN-Water (0.05% AcOH)

Flow rate: 4.0 mL/min

Eluate:

-   -   MeCN/Water 25% (iso, 2 min), then    -   MeCN/Water 25% to 60% (gradient, 20 min), then    -   MeCN/Water 60% (iso, 1 min)

¹H NMR (500 MHz, METHANOL-d4) δ ppm 0.99 (d, J=6.7 Hz, 6H) 1.00-1.03 (m,1H) 1.06 (d, J=6.7 Hz, 3H) 1.10 (d, J=6.1 Hz, 3H) 1.26-1.64 (m, 11H)1.65-2.37 (m, 25H) 2.40 (dd, J=13.4, 6.1 Hz 1H) 2.45 (dd, J=17.1, 1.8Hz, 1H) 2.56 (dd, J=17.7, 9.2 Hz, 1H) 2.76-2.84 (m, 1H) 2.98 (dd, J=9.8,1.8 Hz, 1H) 3.01-3.17 (m, 2H) 3.22 (dd, J=6.7, 4.9 Hz, 1H) 3.34 (dd,J=6.1, 4.3 Hz, 1H) 3.48-3.53 (m, 2H) 3.61 (d, J=11.6 Hz 1H) 3.69-3.75(m, 2H) 3.82 (br. s., 1H) 3.85-3.92 (m, 2H) 3.99 (br. s., 1H) 4.04-4.14(m, 4H) 4.18 (dd, J=6.7, 4.2 Hz, 1H) 4.25 (ddd, J=11.6, 4.9, 1.8 Hz, 1H)4.29-4.35 (m, 2H) 4.46 (d, J=11.0 Hz, 1H) 4.58-4.63 (m, 2H) 4.70 (t,J=7.2, 4.6 Hz, 1H) 4.81 (br. s., 1H) 5.02 (br. s., 1H) 5.06 (br. s.,1H). ESI-MS (m/z): 1080.6 [M+H]⁺, 1102.6[M+Na]⁺.

Synthesis of Compound (H-2) Example 29

The title compound H-2 was obtained as an acetic acid salt from compoundF-1:allyl-2-((2R,3R,5S,6S)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-((2S,6S,7S,E)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methyl-4-oxonon-8-en-2-yl)tetrahydro-2H-pyran-2-yl)ethyl)carbamateprepared by the method written in Scheme F, and compound H-1 (CAS No;1353268-98-0) prepared by the method written in Journal of the AmericanChemical Society 2012, 134, 893-896, the entire contents of which isincorporated herein by reference, and based on the procedure describedin Scheme G. Retention time=10.2 min.

HPLC Conditions

Column: YMC Pak Pro C18 (10 mm I.D.×250 mm)

Detection wavelength: 200 nm

Column temperature: room temperature

Mobile phase: MeCN-Water (0.05% AcOH)

Flow rate: 4.0 ml/min

Eluate:

-   -   MeCN/Water 20% (iso, 2 min), then    -   MeCN/Water 20% to 80% (Gradient, 20 min)

¹H NMR (400 MHz, METHANOL-d₄) δ ppm 0.99 (d, J=7.0 Hz, 6H) 1.02-1.04 (m,1H) 1.06 (d, J=7.0 Hz, 3H) 1.11 (d, J=6.6 Hz, 3H) 1.24-1.45 (m, 7H)1.46-1.54 (m, 2H) 1.54-1.61 (m, 2H) 1.62-1.77 (m, 5H) 1.78-1.89 (m, 4H)1.90-2.16 (m, 7H) 2.16-2.36 (m, 9H) 2.39 (dd, J=13.3, 5.9 Hz 2H) 2.55(dd, J=19.9, 3.5 Hz, 1H) 2.75-2.85 (m, 1H) 2.95 (dd, J=9.8, 2.0 Hz, 1H)3.01-3.17 (m, 2H) 3.22 (dd, J=6.4, 4.5 Hz, 2H) 3.46-3.53 (m, 2H) 3.61(d, J=10.5 Hz 1H) 3.67-3.76 (m, 2H) 3.80-3.84 (m, 1H) 3.84-3.92 (m, 2H)3.97-4.01 (m, 1H) 4.03-4.14 (m, 3H) 4.17 (dd, J=4.1, 1.4 Hz, 1H)4.21-4.27 (m, 1H) 4.27-4.34 (m, 2H) 4.39-4.43 (m, 1H) 4.44 (d, J=10.6Hz, 1H) 4.62 (dd, J=7.2, 4.5 Hz, 1H) 4.82 (br. s., 1H) 4.88 (br. s., 1H)5.02 (br. s., 1H) 5.06 (br. s., 1H). ESI-MS (m/z): 1096.6 [M+H]⁺, 1118.6[M+Na]⁺.

Synthesis of Compound (I-2)

Example 30

The title compound I-2 was obtained from compound I-1:(2S,6S,7S,E)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(2-methoxyethyl)tetrahydro-2H-pyran-2-yl)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methylnon-8-en-4-oneobtained by the method written in Scheme I, and compound D-1 (CAS No;157322-23-1), using the procedures described for the preparation ofCompounds D-4, D-5, and D-6 (Example 24, 25, and 26).

¹H NMR (500 MHz, METHANOL-d4) δ ppm 0.98 (d, J=6.8 Hz, 6H) 1.00-1.03 (m,1H) 1.06 (d, J=6.8 Hz, 3H) 1.10 (d, J=5.8 Hz, 3H) 1.29-1.63 (m, 11H)1.65-1.78 (m, 3H) 1.78-2.13 (m, 12H) 2.13-2.36 (m, 9H) 2.39 (dd, J=14.1,5.4 Hz, 1H) 2.45 (d, J=17.1 Hz, 1H) 2.57 (dd, J=17.5, 9.7 Hz, 1H)2.76-2.84 (m, 1H) 2.98 (d, J=10.2 Hz, 1H) 3.19-3.24 (m, 1H) 3.31-3.32(m, 1H) 3.32 (s, 3H) 3.49-3.57 (m, 4H) 3.61 (d, J=11.2 Hz, 1H) 3.68-3.76(m, 2H) 3.79 (br. s., 1H) 3.84-3.92 (m, 2H) 3.98 (br. s., 1H) 4.03-4.15(m, 4H) 4.18 (t, J=5.8 Hz, 1H) 4.25 (d, J=10.2 Hz, 1H) 4.28-4.36 (m, 2H)4.45 (d, J=11.2 Hz, 1H) 4.58-4.65 (m, 2H) 4.70 (br. t, J=5.4 Hz, 1H)4.81 (br. s., 1H) 4.87 (br. s., 1H) 5.02 (br. s., 1H) 5.06 (br. s.,J=1.5 Hz, 1H). ESI-MS (m/z): 1117.6 [M+Na]⁺.

Synthesis of Compound (J-1) Example 31

The title compound J-1 was obtained from compound E-1:(2S,6S,7S,E)-2-((2S,3S,5R,6R)-3,5-bis((tert-butyldimethylsilyl)oxy)-6-(((tert-butyldimethylsilyl)oxy)methyl)tetrahydro-2H-pyran-2-yl)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methylnon-8-en-4-ol(CAS No; 157323-29-0) and compound H-1 (CAS No; 1353268-98-0), based onthe procedure described in Scheme G. Retention time=12.8 min.

HPLC Conditions

Column: YMC Pak Pro C18 (10 mm I.D.×250 mm)

Detection wavelength: 200 nm

Column temperature: room temperature

Mobile phase: MeCN-Water

Flow rate: 4.5 ml/min

Eluate:

-   -   MeCN/Water 35% (iso, 2 min), then    -   MeCN/Water 35% to 99% (Gradient, 20 min)

¹H NMR (500 MHz, METHANOL-d4) δ ppm 0.97 (d, J=2.9 Hz, 3H) 0.99 (d,J=2.9 Hz, 3H) 1.00-1.03 (m, 1H) 1.05 (d, J=7.3 Hz, 3H) 1.11 (d, J=7.3Hz, 3H) 1.30-1.44 (m, 6H) 1.48-1.57 (m, 4H) 1.61-1.76 (m, 4H) 1.79-1.87(m, 3H) 1.91-1.98 (m, 3H) 2.00-2.14 (m, 8H) 2.24-2.35 (m, 5H) 2.37-2.44(m, 6H) 2.55 (dd, J=17.6, 9.8 Hz, 1H) 2.68-2.87 (m, 1H) 2.94 (dd, J=9.8,2.0 Hz, 1H) 3.22 (dd, J=6.6, 4.7 Hz, 1H) 3.22 (dd, J=6.6, 4.7 Hz, 1H)3.26-3.36 (m, 2H) 3.42-3.56 (m, 2H) 3.60 (d, J=11.7 Hz, 1H) 3.65-3.75(m, 3H) 3.79 (br. s., 1H) 3.83-3.93 (m, 2H) 3.98 (d, J=2.4 Hz, 1H)4.02-4.36 (m, 3H) 4.37-4.51 (m, 2H) 4.62 (dd, J=7.6, 4.6, Hz, 1H) 4.82(br. s., 1H) 4.89 (br. s., 1H) 5.01 (br. s., 1H) 5.06 (br. s., 1H).ESI-MS (m/z): 1119.6 [M+Na]⁺.

Synthesis of Compound (K-2)

Example 32

The title compound K-2 was obtained from compound K-1: allyl((2R,4S)-2,4-bis((tert-butyldimethylsilyl)oxy)-4-((2S,4S,5S)-4-((tert-butyldimethylsilyl)oxy)-5-((2S,6S,7S,E)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methyl-4-oxonon-8-en-2-yl)tetrahydrofuran-2-yl)butyl)carbamateprepared by the method written in Scheme K, and compound D-1 (CAS No;157322-23-1), based on the procedure described in Scheme G. Retentiontime=9.5 min.

HPLC Conditions

Column: YMC Pak Pro C18 (10 mm I.D.×250 mm)

Detection wavelength: 200 nm

Column temperature: room temperature

Mobile phase: MeCN-Water (0.05% AcOH)

Flow rate:4 ml/min

Eluate:

-   -   MeCN/Water 25% (iso, 2 min), then    -   MeCN/Water 25% to 80% (Gradient, 20 min)

¹H NMR (500 MHz, METHANOL-d₄) δ ppm 0.96 (d, J=6.8 Hz, 3H) 1.01 (d,J=7.3 Hz, 3H) 1.00-1.03 (m, 1H) 1.05 (d, J=7.3 Hz, 3H) 1.09 (d, J=6.3Hz, 3H) 1.19-1.63 (m, 9H) 1.64-1.76 (m, 4H) 1.77-1.89 (m, 3H) 1.91-2.21(m, 11H) 2.12-2.21 (m, 3H) 2.22-2.44 (m, 7H) 2.44 (dd, J=17.5, 1.9 Hz,1H) 2.55 (dd, J=17.6, 9.3 Hz, 1H) 2.75-2.87 (m, 2H) 2.97 (dd, J=9.8, 2.0Hz, 1H) 3.05 (d, J=11.7 Hz, 1H) 3.21 (dd, J=6.3, 4.4 Hz, 1H) 3.58 (br.s., 1H) 3.60 (d, J=12.2 Hz, 1H) 3.69 (br. s., 1H) 3.73 (d, J=10.2 Hz,1H) 3.77 (br. s., 1H) 3.84-3.90 (m, 2H) 3.98-4.13 (m, 7H) 4.17 (dd,J=6.1, 4.6 Hz, 1H) 4.24 (d, J=11.2 Hz, 1H) 4.29 (d, J=3.9 Hz, 1H) 4.32(dd, J=9.8, 3.9 Hz, 1H) 4.45 (d, J=10.2 Hz, 1H) 4.58-4.62 (m, 2H) 4.69(t, J=4.6 Hz, 1H) 4.80 (s, 1H) 4.84 (s, 1H) 5.02 (br. s., 1H) 5.06 (br.s., 1H)._ESI-MS (m/z): 1110.6. [M+H]⁺, 1132.6 [M+Na]⁺.

Synthesis of Compound (L-6)

Example 33

The title compound L-6 was obtained as an acetic acid salt from compoundL-1 (CAS No; 1353269-09-6) prepared by the method written in Journal ofthe American Chemical Society 2012, 134, 893-896, the entire contents ofwhich is incorporated herein by reference, by the method described inScheme L. Retention time=5.6 min.

HPLC Conditions

Column: YMC Pak Pro C18 (10 mm I.D.×250 mm)

Detection wavelength: 205 nm

Column temperature: room temperature

Mobile phase: MeCN-Water (0.05% AcOH)

Flow rate: 4.7 mL/min

Eluate:

-   -   MeCN/Water 25% (iso, 2 min), then    -   MeCN/Water 25% to 80% (gradient, 20 min), then    -   MeCN/Water 80% (iso, 2 min)

¹H NMR (500 MHz, METHANOL-d₄) δ ppm 0.94-1.03 (m, 7H) 1.05 (d, J=7.3 Hz,3H) 1.10 (d, J=6.8 Hz, 3H) 1.21-2.41 (m, 36H) 2.44 (dd, J=17.6, 2.2 Hz,1H) 2.54 (dd, J=17.6, 9.2 Hz, 1H) 2.75-2.83 (m, 1H) 2.94 (dd, J=9.5, 1.7Hz, 1H) 3.19-3.23 (m, 1H) 3.31-3.33 (m, 1H) 3.53-3.73 (m, 7H) 3.78 (br.s., 1H) 3.84-3.90 (m, 2H) 3.97-4.12 (m, 4H) 4.16 (dd, J=3.9, 1.0 Hz, 1H)4.21 (dd, J=5.6, 3.2 Hz, 1H) 4.21-4.26 (m, 1H) 4.26-4.33 (m, 2H)4.38-4.46 (m, 2H) 4.58-4.63 (m, 2H) 4.81 (br. s., 1H) 4.86-4.87 (m, 1H)5.01 (br. s., 1H) 5.06 (br. s., 1H). ESI-MS (m/z):1126.6 [M+H]⁺, 1148.6[M+Na]⁺.

Synthesis of Compound (M-3)

Example 34

The title compound M-3 was obtained from compound M-1:(2S,6S,7S,E)-2-((2S,3S,5S)-3-((tert-butyldimethylsilyl)oxy)-5-((S)-2,2,3,3,9,9,10,10-octamethyl-4,8-dioxa-3,9-disilaundecan-5-yl)tetrahydrofuran-2-yl)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methylnon-8-en-4-oneprepared by the method written in Scheme M, and Compound D-1 (CAS No;157322-23-1), using the procedures described for the preparation ofCompounds D-4, D-5, and D-6 (Example 24, 25, and 26).

¹H NMR (500 MHz, METHANOL-d4) δ ppm 0.96 (d, J=6.8 Hz, 3H) 1.00-1.03 (m,1H) 1.02 (d, J=7.3 Hz, 3H) 1.06 (d, J=7.3 Hz, 3H) 1.10 (d, J=6.8 Hz, 3H)1.29-1.63 (m, 10H) 1.65-1.78 (m, 5H) 1.79-1.89 (m, 3H) 1.92-2.12 (m, 7H)2.13-2.36 (m, 10H) 2.40 (dd, J=13.4, 6.1 Hz, 1H) 2.45 (dd, J=17.5, 2.4Hz, 1H) 2.56 (dd, J=18.0, 9.7 Hz, 1H) 2.76-2.84 (m, 1H) 2.98 (dd, J=9.7,1.9 Hz, 1H) 3.22 (dd, J=6.3, 4.9 Hz, 1H) 3.31-3.32 (m, 1H) 3.55 (m, 1H)3.61 (d, J=11.7 Hz, 1H) 3.67-3.76 (m, 5H) 3.84-3.91 (m, 2H) 3.95 (sept,J=4.9 Hz, 1H) 4.03-4.11 (m, 4H) 4.12 (dd, J=6.6, 4.1 Hz, 1H) 4.18 (dd,J=6.3, 4.4 Hz, 1H) 4.24 (ddd, J=11.2, 9.0, 2.3 Hz, 1H) 4.28-4.31 (m, 1H)4.32 (dd, J=10.2, 4.4 Hz, 1H) 4.46 (d, J=10.7 Hz, 1H) 4.60 (t, J=3.9 Hz,1H) 4.62 (dd, J=7.3, 3.9 Hz, 1H) 4.70 (t, J=4.4 Hz, 1H) 4.81 (d, J=1.5Hz, 1H) 4.87 (s, 1H) 5.02 (br. s., 1H) 5.06 (d, J=1.5 Hz, 1H). ESI-MS(m/z): 1103.6 [M+Na]⁺.

Synthesis of Compound (N-1) Example 35

The title compound N-1 was obtained as an acetic acid salt from compoundM-2:allyl-((S)-3-((tert-butyldimethylsilyl)oxy)-3-((2S,4S,5S)-4-((tert-butyldimethylsilyl)oxy)-5-((2S,6S,7S,E)-9-iodo-7-((4-methoxybenzyl)oxy)-6-methyl-4-oxonon-8-en-2-yl)tetrahydrofuran-2-yl)propyl)carbamateprepared by the method written in Scheme M, and Compound D-1 (CAS No;157322-23-1), by the same procedure described in Scheme G. Retentiontime=8.4 min.

HPLC Conditions

Column: YMC Pak Pro C18 (10 mm I.D.×250 mm)

Detection wavelength: 200 nm

Column temperature: room temperature

Mobile phase: MeCN-Water (0.05% AcOH)

Flow rate: 4.5 ml/min

Eluate:

-   -   MeCN/Water 25% (iso, 2 min), then    -   MeCN/Water 25% to 80% (Gradient, 20 min)

¹H NMR (500 MHz, METHANOL-d₄) δ ppm 0.97 (d, J=6.8 Hz 3H) 1.01 (m, 1H)1.02 (d, J=7.8 Hz 3H) 1.07 (d, J=7.3 Hz 3H) 1.10 (d, J=6.3 Hz, 3H)1.26-1.41 (m, 6H) 1.42-1.56 (m, 4H) 1.56-1.64 (m, 2H) 1.64-1.78 (m, 4H)1.81-1.88 (m, 3H) 1.97-2.11 (m, 7H) 2.12-2.22 (m, 2H) 2.23-2.37 (m, 9H)2.40 (dd, J=13.2, 3.9 Hz, 1H) 2.45 (dd, J=17.1, 2.4 Hz, 1H) 2.56 (dd,J=17.8, 10.0 Hz 1H) 2.75-2.87 (m, 1H) 2.98 (dd, J=9.3, 1.9 Hz, 1H)3.06-3.14 (m, 2H) 3.22 (dd, J=5.8, 4.4 Hz, 1H) 3.59 (br. s., 1H) 3.61(d, J=11.2 Hz, 1H) 3.68-3.76 (m, 3H) 3.85-3.92 (m, 2H) 3.98-4.03 (m, 1H)4.04-4.14 (m, 4H) 4.16-4.20 (m, 1H) 4.22-4.28 (m, 1H) 4.28-4.35 (m, 2H)4.46 (d, J=11.21 Hz, 1H) 4.58-4.64 (m, 2H) 4.70 (t, J=4.9 Hz 1H) 4.81(br. s., 1H) 4.87 (br. s., 1H) 5.03 (br. s., 1H) 5.07 (br. s., 1H).ESI-MS (m/z): 1080.6 [M+H]⁺, 1102.6 [M+Na]⁺.

PHARMACOLOGICAL TEST EXAMPLES General Information

Natural Halichondrin compounds and modified compounds thereof are knownin the literature (See, e.g., D. Uemura et al. “Norhalichondrin A: AnAntitumor Polyether Macrolide from a Marine Sponge” J. Am. Chem. Soc.,107, 4796 (1985); Marc Litaudon et al. “Antitumor Polyether Macrolides:New and Hemisynthetic Halichondrins from the New Zealand Deep-WaterSponge Lissodendoryx sp.” J Org. Chem., 1997, 62, 1868-1871). However,most of them are not easily available. For example, Dr. Uemura et.al.isolated 12.5 mg of Halichondrin B, 35.0 mg of Norhalichondrin A and17.2 mg of Homohalichondrin A from as much as 600 kg of Halichondriaokadai Kadota (See, e.g., D. Uemura et al. “Norhalichondrin A: AnAntitumor Polyether Macrolide from a Marine Sponge” J. Am. Chem. Soc.,107, 4796 (1985)). Among natural Halichondrin compounds, Halichondrin Bshows the strongest anti-tumor activities against B-16 melanoma cells invitro and is highly active against L-1210 Leukemia in vivo (See, e.g.,D. Uemura et al. “Norhalichondrin A: An Antitumor Polyether Macrolidefrom a Marine Sponge” J. Am. Chem. Soc., 107, 4796 (1985)). HalichondrinC is also active in various in vivo models but unstable in aqueoussolution in comparison with Halichondrin B. Norhalichondrin B is muchweaker than Halichondrin B not only in vitro but also in vivo See, e.g.,D. Uemura et al. “Norhalichondrin A: An Antitumor Polyether Macrolidefrom a Marine Sponge” J. Am. Chem. Soc., 107, 4796 (1985)). Thefollowing pharmacological tests use Halichondrin B (Hali-B) as referencecompounds as needed.

Pharmacological Test Example 1. FaDu Growth Inhibition Assay

In this assay, the growth inhibitory activity of test compounds in ahuman squamous cell carcinoma of the head and neck (SCCHN) cell lineFaDu was measured. FaDu cells were maintained in an RPMI-1640 (Wako PureChemical Industries, Ltd., 187-02021) medium containing 10% fetal bovineserum (FBS: Nichirei, 12D168), and penicillin and streptomycin in a 5%CO₂ incubator (37° C.). To each well of a 96 well plate (Becton,Dickinson and Company, 353219), 75 μL of FaDu cell suspension adjustedto a concentration of 4×10⁴ cells/mL with the culture medium was added,and the cells were incubated overnight in a 5% CO₂ incubator (37° C.).On the next day, 25 μL of Compound (1) or Halichondrin B in three-folddilution series suspended in the culture medium was added to each well,and the resultant was incubated for 3 days in a 5% CO₂ incubator (37°C.). Then, cell viability was determined by CellTiter-Glo® LuminescentCell Viability Assay (Promega) with EnVision 2103 Multilabel Reader(Perkin-Elmer, Wellesley, Mass.). Value of the wells containing cellswithout adding the test compounds was defined as 100% and the value ofthe wells containing no cells was defined as 0%. The concentration ofthe test compound necessary for inhibiting the cell growth by 50% (i.e.,an IC₅₀ value) was calculated, and shown in Table 1.

TABLE 1 FaDu Test compound (IC₅₀ (nM)) Halichondrin B 0.124 Compound (1)0.0714

Pharmacological Test Example 2. MDA-MB231 Growth Inhibition Assay

In this assay, the growth inhibitory activity of test compounds in ahuman breast cancer cell line MDA-MB231 was measured. MDA-MB231 cellswere maintained in Dulbecco's Modified Eagle's medium (Wako PureChemical Industries, Ltd., 044-29765) medium containing 10% fetal bovineserum (FBS: Nichirei, 12D168), and penicillin and streptomycin in a 5%CO₂ incubator (37° C.). To each well of a 96 well plate (Becton,Dickinson and Company, 353219), 75 μL of MDA-MB231 cell suspensionadjusted to a concentration of 4×10⁴ cells/mL with the culture mediumwas added, and the cells were incubated overnight in a 5% CO₂ incubator(37° C.). On the next day, 25 μL of Compound (1) or Halichondrin B inthree-fold dilution series suspended in the culture medium was added toeach well, and the resultant was incubated for 3 days in a 5% CO₂incubator (37° C.). Then, cell viability was determined byCellTiter-Glo® Luminescent Cell Viability Assay (Promega) with EnVision2103 Multilabel Reader (Perkin-Elmer, Wellesley, Mass.). Value of thewells containing cells without adding the test compounds was defined as100% and the value of the wells containing no cells was defined as 0%.The concentration of the test compound necessary for inhibiting the cellgrowth by 50% (i.e., an IC₅₀ value) was calculated, and shown in Table2.

TABLE 2 MDA-MB231 Test compound (IC₅₀ (nM)) Halichondrin B 1.000Compound (1) 0.109

Pharmacological Test Example 3. HCC1954 Growth Inhibition Assay

In this assay, the growth inhibitory activity of test compounds in ahuman breast cancer cell line HCC1954 was measured. HCC1954 cells weremaintained in an RPMI-1640 medium modified to contain 2 mM L-glutamine,10 mM HEPES, 1 mM sodium pyruvate, 4500 mg/L glucose, and 1500 mg/Lsodium bicarbonate (ATCC 30-2001) containing 10% fetal bovine serum(FBS: Nichirei, 12D168), and penicillin and streptomycin in a 5% CO₂incubator (37° C.). To each well of a 96 well plate (Becton, Dickinsonand Company, 353219), 75 μL of HCC1954 cell suspension adjusted to aconcentration of 4×10⁴ cells/mL with the culture medium was added, andthe cells were incubated overnight in a 5% CO₂ incubator (37° C.). Onthe next day, 25 μL of Compound (1) or Halichondrin B in three-folddilution series suspended in the culture medium was added to each well,and the resultant was incubated for 3 days in a 5% CO₂ incubator (37°C.). Then, cell viability was determined by CellTiter-Glo® LuminescentCell Viability Assay (Promega) with EnVision 2103 Multilabel Reader(Perkin-Elmer, Wellesley, Mass.). Value of the wells containing cellswithout adding the test compounds was defined as 100% and the value ofthe wells containing no cells was defined as 0%. The concentration ofthe test compound necessary for inhibiting the cell growth by 50% (i.e.,an IC₅₀ value) was calculated, and shown in Table 3.

TABLE 3 HCC1954 Test compound (IC₅₀ (nM)) Halichondrin B 0.154 Compound(1) 0.0668

Pharmacological Test Example 4. Antitumor Effects in FaDu SubcutaneousXenograft Model in Mice as Monotherapy

A human squamous cell carcinoma of the head and neck (SCCHN) cell lineFaDu, which had been cultured in an RPMI-1640 medium containing 10% FBS,and penicillin and streptomycin, was adjusted to a concentration of4.8×10⁷ cells/mL with Hanks' Balanced Salt Solution to prepare a cellsuspension. The cell suspension was inoculated in a volume of 100 μLinto a subcutaneous part of a right flank of nude mice, 7 weeks of ages(CAnN.Cg-Foxnlnu/CrlCrlj, female, Charles River Laboratories JapanInc.). Nine days after cell inoculation, the shortest diameter and thelongest diameter of a tumor in each mouse were measured by using anelectronic digital caliper (Digimatic™ caliper, Mitutoyo Corporation),so as to calculate the volume of the tumor in accordance with thefollowing calculation formulae:

Tumor volume (mm³)=Longest diameter (mm)×Shortest diameter (mm)×Shortestdiameter (mm)/2

Relative tumor volume (RTV)=Tumor volume (day X)/Tumor volume (the firstday) Tumor Regression (%)=(1−minimum RTV)×100

On the basis of the volumes of tumors obtained on the first day ofadministration, the mice were grouped such that averages of the tumorvolumes were substantially equal among the groups. Each test compoundwas dissolved in DMSO and a solution was stored at the freezer beforeuse. Immediately before the administration, the stock solution wasdiluted in saline with 100 μM of hydroxypropyl-β-cyclodextrin. Eachevaluation sample was intravenously administered at a maximum tolerabledose (MTD). Incidentally, the experiment was conducted on groups eachconsisting of 4 mice. Tumor regression (%) of each test compound wasshown in Table 4.

TABLE 4 Dose Tumor Regression Test compound (mg/kg) (%) Halichondrin B0.05 0 Compound (1) 0.2 43

Pharmacological Test Example 5. Antitumor Activity Against OSC-19 inSubcutaneous Xenograft Model in Mice as Monotherapy

A human squamous cell carcinoma of the head and neck (SCCHN) cell lineOSC-19, which had been cultured in an DMEM/Ham's F-12 (1:1) mediumcontaining 10% FBS, and penicillin and streptomycin, was adjusted to aconcentration of 1×10⁸ cells/ml with PBS to prepare a cell suspension,and the suspension was mixed with Matrigel™ (BD Bioscience, #366237) ina ratio of 1:1 to prepare a cell suspension in a concentration of 5×10⁷cell/mL. The cell suspension was inoculated in a volume of 100 μL into asubcutaneous part of a right flank of nude mice, 5 weeks of ages(CAnN.Cg-Foxnlnu/CrlCrlj, female, Charles River Laboratories Japan,Inc.). Six days after cell inoculation, the shortest diameter and thelongest diameter of a tumor in each mouse were measured by using anelectronic digital caliper (Digimatic™ caliper, Mitutoyo Corporation),so as to calculate the volume of the tumor in accordance with thefollowing calculation formulae:

Tumor volume (mm³)=Longest diameter (mm)×Shortest diameter (mm)×Shortestdiameter (mm)/2

Relative tumor volume (RTV)=Tumor volume (day X)/Tumor volume (the firstday) Tumor Regression (%)=(1−minimum RTV)×100

On the basis of the volumes of tumors obtained on the first day ofadministration, the mice were grouped such that averages of the tumorvolumes were substantially equal among the groups. The experiment wasconducted on groups each consisting of 6 mice. Test compound wasdissolved in saline and intravenously administered at doses from 0.06mg/kg to 0.18 mg/kg once a week for 2 weeks (Q7Dx2 schedule). Tumorregression (%) of each test dose is shown in Table 5.

TABLE 5 Dose Tumor Regression Test compound (mg/kg) (%) Compound (1)0.06 59 Compound (1) 0.18 90

Pharmacological Test Example 6. Antitumor Activity Against HCC1806 inSubcutaneous Xenograft Model in Mice as Monotherapy

A human breast cancer cell line HCC1806, which had been cultured in anRPMI-1640 medium containing 10% FBS, and penicillin and streptomycin,was adjusted to a concentration of 1×10⁸ cells/mL with PBS to prepare acell suspension, and the suspension was mixed with Matrigel™ (BDBioscience, #366237) in a ratio of 1:1 to prepare a cell suspension in aconcentration of 5×10⁷ cell/mL. The cell suspension was inoculated in avolume of 100 μL into a subcutaneous part of a right flank of nude mice,5 weeks of ages (CAnN.Cg-Foxnlnu/CrlCrlj, female, Charles RiverLaboratories Japan, Inc.). Twelve days after cell inoculation, theshortest diameter and the longest diameter of a tumor in each mouse weremeasured by using an electronic digital caliper (Digimatic™ caliper,Mitutoyo Corporation), so as to calculate the volume of the tumor inaccordance with the following calculation formulae:

Tumor volume (mm³)=Longest diameter (mm)×Shortest diameter (mm)×Shortestdiameter (mm)/2

Relative tumor volume (RTV)=Tumor volume (day X)/Tumor volume (the firstday) Tumor Regression (%)=(1−minimum RTV)×100

On the basis of the volumes of tumors obtained on the first day ofadministration, the mice were grouped such that averages of the tumorvolumes were substantially equal among the groups. The experiment wasconducted on groups each consisting of 6 mice. Test compound wasdissolved in saline and intravenously administered at 0.18 mg/kg once aweek for 2 weeks (Q7Dx2 schedule). Tumor regression (%) for Compound (1)is shown in Table 6.

TABLE 6 Dose Tumor Regression Test compound (mg/kg) (%) Compound (1)0.18 90

Pharmacological Test Example 7. Antitumor Effects in FaDu SubcutaneousXenograft Model in Combination with Cetuximab in Mice

A human squamous cell carcinoma of the head and neck (SCCHN) cell lineFaDu, which had been cultured in an RPMI-1640 medium containing 10% FBS,and penicillin and streptomycin, was adjusted to a concentration of5×10⁷ cells/mL with Hanks' Balanced Salt Solution to prepare a cellsuspension. The cell suspension was inoculated in a volume of 100 L intoa subcutaneous part of a right flank of nude mice, 7 weeks of ages(CAnN.Cg-Foxnlnu/CrlCrlj, female, Charles River Laboratories JapanInc.). Ten days after cell inoculation, the shortest diameter and thelongest diameter of a tumor in each mouse were measured by using anelectronic digital caliper (Digimatic™ caliper, Mitutoyo Corporation),so as to calculate the volume of the tumor in accordance with thefollowing calculation formulae:

Tumor volume (mm³)=Longest diameter (mm)×Shortest diameter (mm)×Shortestdiameter (mm)/2

Relative tumor volume (RTV)=Tumor volume (day X)/Tumor volume (the firstday) Tumor Regression on day 35(%)=(1−RTV on day 35)×100

On the basis of the volumes of tumors obtained on the first day ofadministration, the mice were grouped such that averages of the tumorvolumes were substantially equal among the groups. Each test compoundwas dissolved in DMSO and a solution was stored at the freezer beforeuse. Immediately before the administration, the stock solution wasdiluted in saline with 100 μM of hydroxypropyl-o-cyclodextrin. Each testcompound and was intravenously administered at doses from ¼ MTD to ½ MTDin combination with cetuximab (Erbitux, Merck Serono Co., Ltd.).Incidentally, the experiment was conducted on groups each consisting of4 mice. Tumor regression on day 35(%) of each test compound are shown inTable 7.

TABLE 7 Tumor Regression Dose Cetuximab on day 35 Test compound (mg/kg)(mg/kg) (%) — — 20 −242 Halichondrin B 0.0125 20 −38 0.025 20 −2Compound (1) 0.05 20 38 0.1 20 60

Pharmacological Test Example 8. Antitumor Activity in KPL-4 SubcutaneousXenograft Model in Combination with Trastuzumab in Mice

A human HER-2 positive breast cancer cell line KPL-4, which had beencultured in an RPMI-1640 medium containing 10% FBS, and penicillin andstreptomycin, was adjusted to a concentration of 1×10⁸ cells/mL withHank's Balanced Salt Solution to prepare a cell suspension. The cellsuspension was inoculated in a volume of 100 μL into a subcutaneous partof a right flank of nude mice, 7 weeks of ages (CAnN.Cg-Foxnlnu/CrlCrlj,female, Charles River Laboratories Japan, Inc.). Sixteen days after thecell inoculation, the shortest diameter and the longest diameter of atumor in each mouse were measured by using an electronic digital caliper(Digimatic™ caliper, Mitutoyo Corporation), so as to calculate thevolume of the tumor in accordance with the following calculationformulae:

Tumor volume (mm³)=Longest diameter (mm)×Shortest diameter (mm)×Shortestdiameter (mm)/2

Relative tumor volume (RTV)=Tumor volume (day X)/Tumor volume (the firstday) Tumor Regression (%)=(1−minimum RTV)×100

On the basis of the volumes of tumors obtained on the first day ofadministration, the mice were grouped such that averages of the tumorvolumes were substantially equal among the groups. The experiment wasconducted on groups each consisting of 6 mice. Each test compound wasdissolved in DMSO and a solution was stored at the freezer before use.

Immediately before the administration, the stock solution was diluted insaline. The test compound was intravenously administered at 0.09 mg/kgor 0.18 mg/kg in combination with trastuzumab (Herceptin, Genentech,Inc.). Tumor regression for Compound (1) is shown in Table 8.

TABLE 8 Dose Trastuzumab Tumor Regression Test compound (mg/kg) (mg/kg)(%) — — 10 0 Compound (1) 0.09 — 43 0.09 10 83 0.18 — 87 0.18 10 100

Pharmacological Test Example 9. Effect on CD31-Positive Vessel in theFaDu Subcutaneous Model in Mice

A human squamous cell carcinoma of the head and neck (SCCHN) cell lineFaDu, which had been cultured in an RPMI-1640 medium containing 10% FBS,and penicillin and streptomycin, was adjusted to a concentration of5×10⁷ cells/mL with PBS to prepare a cell suspension. The cellsuspension was inoculated in a volume of 100 μL into a subcutaneous partof a right flank of nude mice, 7 weeks of ages (CAnN.Cg-Foxnlnu/CrlCrlj,female, Charles River Laboratories Japan, Inc.). Ten days after cellinoculation, a test compound in saline with 100 μM ofhydroxypropyl-o-cyclodextrin was intravenously administered at dosesfrom ½ MTD to MTD. The experiment was conducted on groups eachconsisting of 3 mice. Five days after administration, tumor samples werecollected and fixed with IHC Zinc Fixative (BD Pharmingen) at 4° C. for24 hr. Paraffin-embedded tissues were sectioned (3 μm), mounted onpositively charged slides, and air-dried. Immunohistochemical stainingof CD31 was conducted using Ventana autostainer model Discover XT (RocheDiagnostics) according to the manufacture's protocol. Sections weredeparaffinized, conditioned and the antigens were retrieved with CC1(Ventana Medical Systems). Slides were blocked with Blocker A andBlocker B (Endogenous biotin blocking kit, Roche Diagnostics). Ratanti-mouse IgG CD31 antibody (Dianova GmbH) was applied at 2 μg/mL.Sections were incubated with the antibody for 6 hr, followed by 32minutes incubation with biotinylated anti-rat IgG antibody (JacksonImmunoResearch Laboratories) at 2.2 μg/mL. The detection was performedwith Streptavidin-HRP D for 16 min, followed by incubation with DAB Dand DAB H₂O₂ D (DABMap kit, Ventana Medical Systems, Inc) for 8 min.Slides were counterstained with Hematoxylin II (Roche Diagnostics) for16 min, followed by incubation with Bluing reagent for 4 min. Sectionswere dehydrated in graded ethanols, defatted in xylene replacement andcovered with DPX (Merck KGaA).

Immunostained slides were scanned using Vectra 2 Automated Slide ImagingSystem (Perkin Elmer Inc.). The number of blood vessels in the wholetumor was quantified by counting the CD31-positive objects using inForm2 software (PerkinElmer Inc.) Area of the tumor region was measured byassessing the hematoxylin-staining area using inform 2 software(PerkinElmer Inc.) The number of blood vessels was normalized by thearea of the tumor region. An increase rate of the blood vessel number ofthe test compound-dosing group was calculated with the below formula,and shown in Table 9.

Increase rate of blood vessel number (%)=((blood vessel number of thetest compound-dosing group−blood vessel number of the controlgroup)/blood vessel number of the control group)×100

TABLE 9 Increase rate of Dose blood vessel number Test compound (mg/kg)(%) Halichondrin B 0.025 31 0.05 39 Compound (1) 0.10 69 0.20 154

Pharmacological Test Example 10. Effect on α-SMA Positive-CAFs in theFaDu Subcutaneous Model

A human squamous cell carcinoma of the head and neck (SCCHN) cell lineFaDu, which had been cultured in an RPMI-1640 medium containing 10% FBS,and penicillin and streptomycin, was adjusted to a concentration of5×10⁷ cells/mL with PBS to prepare a cell suspension. The cellsuspension was inoculated in a volume of 100 μL into a subcutaneous partof a right flank of nude mice, 5 to 6 weeks of ages(CAnN.Cg-Foxnlnu/CrlCrlj, female, Charles River Laboratories Japan,Inc.). Ten days after cell inoculation, a test compound in saline with100 μM of hydroxypropyl-o-cyclodextrin was intravenously administered at½ MTD and MTD. The experiment was conducted on groups each consisting of3 mice. Two days after administration, tumor samples were collected andfixed with TIC Zinc Fixative (BD Pharmingen) at 4° C. for 24 hr.Paraffin-embedded tissues were sectioned (3 m), mounted on positivelycharged slides, and air-dried for 6 hr.

Immunohistochemical staining of α-SMA was conducted using Ventanaautostainer model Discover XT (Roche Diagnostics). Sections weredeparaffinized, conditioned and the antigens were retrieved withproprietary buffers, EZPrep and CC1 (Ventana Medical Systems). Mouseanti-α-SMA monoclonal antibody conjugated with alkaline phosphatase(clone 1A4, Sigma) was applied at 5 μg/mL. Sections were incubated withthe antibody for 6 hr. The detection was performed with RedMap kit(Ventana Medical Systems, Inc). Sections were dehydrated in gradedethanols, defatted in xylene replacement and covered with DPX (MerckKGaA). The serial tumor slices were deparaffinized and stained withMayer's hematoxylin (Muto Pure Chemicals) for 1 min. Sections weredehydrated in graded ethanols, defatted in xylene replacement andcovered with DPX (Merck KGaA).

Immunostained slides were scanned using Vectra 2 Automated Slide ImagingSystem (Perkin Elmer Inc.). The area of the α-SMA-positive region in thewhole tumor was quantified by counting the α-SMA-positive objects usinginForm 2 software (PerkinElmer Inc.). Area of the tumor region wasmeasured by assessing the hematoxylin-staining area using inForm 2software (PerkinElmer Inc.). The area of the α-SMA positive region wasnormalized by the area of the tumor region. A suppression rate of theα-SMA positive area of the test compound-dosing group was calculatedwith the below formula, and shown in Table 10.

TABLE 10 Suppression rate of Dose α-SMA positive area Test compound(mg/kg) (%)^(a) Halichondrin B 0.025 7 0.05 3 Compound (1) 0.10 21 0.2028 ^(a)Suppression rate of α-SMA positive area (%) = −((α-SMA positivearea of the test compound-dosing group − α-SMA positive area of thecontrol group)/α-SMA positive area of the control group) × 100

Pharmacological Test Example 11. HSC-2 Orthotopic Transplantation MouseModel

Luciferase-transduced HSC-2-Luc cells were established byretrovirus-mediated gene transfer. First, the DNA fragment encodingfirefly luciferase was obtained from pGL3-enhancer plasmid (GenBank#:U47297), and subcloned into the retroviral vector pCX4pur (GenBank #:AB086386). Then, helper-free recombinant retroviruses were produced bytransfecting the above retroviral expression vector together with pGPand pE-Ampho plasmids (Takara Bio; Shiga, Japan), into 293T cells (ATCC;Manassas, USA). Next, HSC-2 cells were infected with the recombinantretroviruses, and were cultured for two weeks in the presence ofpuromycin (2 μg/mL). The infected cells were selected from a polyclonalproliferative population of the culture.

Under anesthesia, the human SCCHN cell line, HSC-2-Luc was inoculatedinto tongue of female nude mice (1×10⁶ cells in 50 μL of PBS), 6 weeksof ages (CAnN.Cg-Foxnlnu/CrlCrlj mice; Charles River, Inc.; Shizuoka,Japan). Seven days after transplantation, the tumor volume was analyzedusing bioluminescence signal from HSC-2-Luc cells. For bioluminescenceimaging, 0.1 mL of 15 mg/mL D-luciferin (Promega, Madison, Wis.) wasinjected intraperitoneally into nude mice under 1% to 2% inhaledisoflurane anesthesia. The bioluminescence signal was monitored usingthe IVIS SPECTRUM series (PerkinElmer, Waltham, Mass.), consisting of ahighly sensitive, cooled chargecoupled device camera. Living Imagesoftware (PerkinElmer, Waltham, Mass.) was used to grid the imaging dataand integrate the total bioluminescence signal in eachregion-of-interest (ROI). All bioluminescence images were acquired witha 1 second exposure. Data were analyzed using total photon flux emission(photons/second) in the ROIs.

On the basis of the total photon flux emission obtained on the first dayof administration, the mice were grouped such that averages of the totalphoton flux emission were substantially equal among the groups. Compound(1) or cisplatin was intravenously administered with or withoutcetuximab (Erbitux, Merck Serono Co., Ltd.) once a week for 3 weeks(Q7Dx3 schedule). Two experiments were conducted using the identicalprocedure and all data were collected from the experiments. Each groupconsisted of 16 mice.

The imaging data showed that only the treatment of Compound (1) withcetuximab clearly reduced the bioluminescence signal in all mice afterDay 14 (FIG. 6A-6B). Median survival time (MST) was calculated for eachgroup of treatment as the median of the days of death. Increase LifeSpan (ILS) was calculated by the following formula: ILS (%)=(MST ofanimals treated with test compound−MST of control animals)/MST ofcontrol animals×100. ILS (%) of each test compound is shown in Table 11.

TABLE 11 Dose Cetuximab ILS Test compound (mg/kg) (mg/kg) (%) — — 5 231Cisplatin 5 — 0 5 5 150 Compound (1) 0.09 — 238 0.09 5 >1150

Pharmacological Test Example 12. FaDu s.c. Xenograft Model inCombination with Radiation

Luciferase-transduced FaDu-Luc cells were established byretrovirus-mediated gene transfer. First, the DNA fragment encodingfirefly luciferase was obtained from pGL3-enhancer plasmid (GenBank#:U47297), and subcloned into the retroviral vector pCX4pur (GenBank #:AB086386). Then, helper-free recombinant retroviruses were produced bytransfecting the above retroviral expression vector together with pGPand pE-Ampho plasmids (Takara Bio; Shiga, Japan), into 293T cells (ATCC;Manassas, USA). Next, FaDu cells were infected with the recombinantretroviruses, and were cultured for two weeks in the presence ofpuromycin (2 μg/mL). The infected cells were selected from a polyclonalproliferative population of the culture.

A luciferase-transduced human SCCHN cell line FaDu-Luc, which had beencultured in an RPMI-1640 medium containing 10% FBS, and penicillin andstreptomycin, was adjusted to a concentration of 5×10⁷ cells/mL withHank's Balanced Salt Solution to prepare a cell suspension. The cellsuspension was inoculated in a volume of 100 μL into a subcutaneous partof a right thigh of nude mice, 6 weeks of ages (CAnN.Cg-Foxnlnu/CrlCrlj,female, Charles River Laboratories Japan, Inc.). Thirteen days after thecell inoculation, the tumor volume was analyzed using bioluminescencesignal from FaDu-Luc cells. For bioluminescence imaging, 0.1 mL of 15mg/mL D-luciferin (Promega, Madison, Wis.) was injectedintraperitoneally into nude mice under 1% to 2% inhaled isofluraneanesthesia. The bioluminescence signal was monitored using the IVISSPECTRUM series (PerkinElmer, Waltham, Mass.), consisting of a highlysensitive, cooled chargecoupled device camera. Living Image software(PerkinElmer, Waltham, Mass.) was used to grid the imaging data andintegrate the total bioluminescence signal in each region-of-interest(ROI). All bioluminescence images were acquired with a 1 secondexposure. Data were analyzed using total photon flux emission(photons/second) in the ROIs. The total photon flux emission wascalculated in accordance with the following calculation formulae:

Relative bioluminescence level=Total photon flux emission (day X)/Totalphoton flux emission (the first day)

Tumor Regression (%)=(1−minimum Relative bioluminescence level)×100

On the basis of the total photon flux emission obtained on the first dayof administration, the mice were grouped such that averages of the totalphoton flux emission were substantially equal among the groups. Theexperiment was conducted on groups each consisting of 6 mice. Compound(1) was administrated via tail vein injection on day 1 and 8.Irradiation was performed 18 Gy on day 4 and day 11. Tumor regressionfor Compound (1) is shown in Table 12.

TABLE 12 Dose Radiation Tumor Regression Test compound (mg/kg) (Gy) (%)— — 18 16 Compound (1) 0.09 — 0 0.09 18 97

Pharmacological Test Example 13. Antitumor Activity in CT26 SubcutaneousSyngeneic Model in Combination with Anti-mPD-1 Antibody in Mice

A murine undifferentiated colon carcinoma cell line CT26, which had beencultured in an RPMI-1640 medium containing 10% FBS, and penicillin andstreptomycin, was adjusted to a concentration of 2×10⁷ cells/mL withHank's Balanced Salt Solution to prepare a cell suspension. On day 1,the cell suspension was inoculated in a volume of 100 μL into asubcutaneous part of a right flank of BALB/c mice, 6 weeks of ages(BALB/cAnNCrlCrlj, female, Charles River Laboratories Japan, Inc.). Twodays after the cell inoculation, the mice were randomly divided intofour groups and each group consists of 8 mice. The shortest diameter andthe longest diameter of a tumor in each mouse were measured by using anelectronic digital caliper (Digimatic™ caliper, Mitutoyo Corporation),so as to calculate the volume of the tumor in accordance with thefollowing calculation formula:

Tumor volume (mm³)=Longest diameter (mm)×Shortest diameter (mm)×Shortestdiameter (mm)/2

T/C=(mean tumor volume of treated group)/(mean tumor volume of controlgroup) Inhibition of tumor growth (%)=(1−T/C)×100

The test compound was intravenously administered at 0.09 mg/kg on days 3and 11. Anti-mPD-1 antibody (BE0146, Bio X Cell) was intravenouslyadministered at 10 mg/kg on days 3, 7, 11, and 15. Inhibition of tumorgrowth on day 15(%) of each test compound is shown in Table 13.

TABLE 13 Anti-mPD-1 Inhibition of tumor Dose antibody growth on day 15Test compound (mg/kg) (mg/kg) (%) — — 10 32 Compound (1) 0.09 — 30 0.0910 62

Pharmacological Test Example 14. Effect on Tubulin Polymerization InVitro (FIG. 10A)

Tubulin Polymerization Assay kit was purchased from Cytoskeleton, Inc.(Cat. #BK011P). The kit contained 1 bottle of lyophilized tubulinprotein purified from porcine brain, 3 tubes of lyophilized GTP, 2bottles of lyophilized assay buffer, and 1 bottle of tubulin glycerolbuffer. Assay buffer was prepared by dissolving the contents in 10 mL ofdeionized and sterilized water. This solution contained 80 mmol/Lpiperazine-N,N′-bis[2-ethanesulfonic acid] sesquisodium salt, 2.0 mmol/Lmagnesium chloride, 0.5 mmol/L ethylene glycol-bis(2-amino-ethyl ether)N,N,N′,N′-tetra-acetic acid, pH 6.9, and 10 μmol/L fluorescent reporter.The buffer was stored at −70° C. until use. Tubulin glycerol bufferconsisted of 80 mmol/L piperazine-N,N′-bis[2-ethanesulfonic acid]sesquisodium salt, 2.0 mmol/L magnesium chloride, 0.5 mmol/L ethyleneglycol-bis(2-amino-ethyl ether) N,N,N′,N′-tetra-acetic acid, and 60% v/vglycerol, pH 6.9. It was stored at 4° C. until use. GTP stock solutionwas prepared by dissolving the contents of each tube in 100 μL ofdeionized and sterilized water to achieve a concentration of 100 mmol/LGTP. Aliquots of this stock were stored at −70° C. until use. Tubulinstock solution (10 mg/mL) was prepared by dissolving the tubulin powderadding 1.1 mL of the mixture of assay buffer and GTP stock solution(100:1, v/v). Aliquots were frozen in liquid nitrogen, and then storedat −70° C. until use.

In the tubulin polymerization assay, reaction mixture was prepared bymixing 820 μL of assay buffer, 17.6 μL of GTP stock solution, and 600 μLof tubulin glycerol buffer. Reaction mixture (1015 μL) was combined with240 μL of the tubulin stock solution. This solution was called astubulin reaction mixture and used for the measurement of test andcontrol wells. No tubulin reaction mixture was prepared by mixing 89.85μL of reaction mixture and 21.25 μL of assay buffer for the measurementof blank wells. The Compound (1) solution (6.25-100 μmol/L; finalconcentrations 0.625-10 μmol/L), or vehicle was added at 5 μL toindividual wells of a 96-well half-area microtiter plate. Tubulinreaction mixture or no tubulin reaction mixture was added at 45 μL toeach well of the plate. Fluorescence emission at 460 nm (excitationwavelength at 360 nm) was measured every 2 minutes for 90 minutes usingthe SpectraMax® M5e microplate reader (Molecular Devices). Tubulinpolymerization was followed by fluorescence enhancement due to theincorporation of a fluorescence reporter into microtubules aspolymerization occurred. The assay was performed in duplicate.

The assay demonstrated that Compound (1) inhibited tubulinpolymerization in a concentration-dependent manner. The fluorescenceintensity in each time point was calculated by the following formulas:

Fluorescence intensity=mean fluorescence measurement of test wells orcontrol wells−mean fluorescence measurement of blank wells; blank well:with vehicle without tubulin; control well: with vehicle and tubulin;test well: with compounds and tubulin.

Pharmacological Test Example 15. Cell-Based Microtubule Dynamics Assay(FIG. 10B)

A cell-based microtubule (MT) dynamics assay was conducted with theU2OS-EB3-AG osteosarcoma cell line, in which the fusion protein of EB3(a microtubule plus end binding protein) and Azami-Green (EB3-AG) wasstably expressed. U2OS-EB3-AG cells were culture in RPMI-1640 mediumcontaining 10% FBS, and penicillin-streptomycin, at 37° C. in ahumidified 5% CO₂ atmosphere. The MT dynamics in the live cells can bevisualized as the movement of the comet-like structure of EB3-AG.U2OS-EB3-AG cells plated on glass-base culture plates (EZVIEW plate, AGCTechno Glass, Japan) were treated with Compound (1) at the indicatedconcentration and the microtubule dynamics were monitored by time-lapseimaging using fluorescent microscope with 60-fold magnificationoil-immersed objective lens (BZ-X710, KEYENCE, Japan). The still imagesat the indicated time points were presented in FIG. 10B. Highermagnification views of the boxed areas were shown in inlets. Whentreated with Compound (1) at 0.5 nM (IC₅₀ value for antiproliferativeactivity in U20S-EB3-AG cells), the comet-like structures became hard toobserve at around 60 minutes after the addition of the compound. Theseresults clearly demonstrated that Compound (1) had the ability tosuppress the MT dynamics.

Pharmacological Test Example 16. In Vitro Antiproliferative Activity(FIG. 11)

The in vitro antiproliferative assays for Compound (1) were conductedusing a small panel of cancer cell lines including human squamous cellcarcinoma of the esophagus (OE21, TE-8), human adenosarcoma of theesophagus (OE33), and human uterine sarcoma (MES-SA, MES-SA-Dx5-Rx1).All cell lines were cultured in RPMI-1640 medium containing 10% FBS, andpenicillin-streptomycin (culture medium), in a 5% CO₂ incubator (37°C.). To each well of a 96 well plate (Becton, Dickinson and Company,353219), 75 μL of cell suspension adjusted to a concentration of 4×10⁴cells/mL with the culture medium was added, and the cells were incubatedovernight in a 5% CO₂ incubator (37° C.). On the next day, 25 μL ofCompound (1) in three-fold dilution series suspended in the culturemedium was added to each well, and the resultant was incubated for 72hours in a 5% CO₂ incubator (37° C.). Then, cell viability wasdetermined by CellTiter-Glo® Luminescent Cell Viability Assay (Promega)with 2013 EnVision™ Multilabel Reader (Perkin-Elmer, Wellesley, Mass.).Value of the wells containing cells without adding the test compoundswas defined as 100% and the value of the wells containing no cells wasdefined as 0%. The concentration of Compound (1) necessary forinhibiting the cell growth by 50% (i.e., an IC₅₀ value) was calculate,and is shown in FIG. 11 . The P-gp susceptibility was calculated as theratio of the IC₅₀ value in MES-SA-Dx5-Rx1 cells, which overexpress P-gp,to the IC₅₀ value in MES-SA cells.

Pharmacological Test Example 17. Antitumor Effects in the KPL-4Xenograft Models in Mice as Monotherapy; Antitumor Effects in theCOLO-704 Xenograft Models in Mice as Monotherapy (FIG. 12)

A human HER-2 positive breast cancer cell line KPL-4, which had beencultured in a DMEM containing 10% FBS, and penicillin-streptomycin, wasadjusted to a concentration of 1×10⁸ cells/mL with Hanks' Balanced SaltSolution to prepare a cell suspension. The cell suspension wasinoculated in a volume of 100 μL into a subcutaneous part of a rightflank of nude mice, 8 weeks of ages (CAnN.Cg-Foxnlnu/CrlCrlj, female,Charles River Laboratories Japan Inc.). Eleven days after cellinoculation (Day 1), the shortest diameter and the longest diameter of atumor in each mouse were measured by using an electronic digital caliper(Digimatic™ caliper, Mitutoyo Corporation), so as to calculate thevolume of the tumor in accordance with the following calculationformulae:

Tumor volume (mm³)=Longest diameter (mm)×Shortest diameter (mm)×Shortestdiameter (mm)/2

Relative tumor volume (RTV)=Tumor volume (day X)/Tumor volume (the firstday)

Relative body weight (RBW)=Body weight (day X)/Body weight (the firstday)

On the basis of the volumes of tumors obtained on Day 1, the mice weregrouped such that averages of the tumor volumes were substantially equalamong the groups. The experiment was conducted on groups each consistingof six mice. The test compound was dissolved in DMSO and a solution wasstored in the freezer before use. Immediately before the administration,the stock solution was diluted with saline. The test compound in salinewas intravenously once-weekly administered at 20 μg/kg, 60 μg/kg, or 180μg/kg for 2 weeks (on Day 1 and Day 8). The tumor regression wasobserved in 60 μg/kg- and 180 μg/kg-treated groups, and theadministration at 180 μg/kg completely eradicated the xenograft tumorsin all mice on Day 15.

A human ovarian cancer cell line COLO-704, which had been cultured in aRPMI-1640 containing 10% FBS, and penicillin-streptomycin, was adjustedto a concentration of 1×10⁸ cells/mL with Hanks' Balanced Salt Solutionto prepare a cell suspension. The cell suspension was inoculated in avolume of 100 μL into a subcutaneous part of a right flank of nude mice,5 weeks of ages (CAnN.Cg-Foxnlnu/CrlCrlj, female, Charles RiverLaboratories Japan Inc.). Nine days after cell inoculation (Day 1), theshortest diameter and the longest diameter of a tumor in each mouse weremeasured by using an electronic digital caliper (Digimatic™ caliper,Mitutoyo Corporation), so as to calculate the volume of the tumor inaccordance with the following calculation formulae:

Tumor volume (mm³)=Longest diameter (mm)×Shortest diameter (mm)×Shortestdiameter (mm)/2

Relative tumor volume (RTV)=Tumor volume (day X)/Tumor volume (the firstday)

Relative body weight (RBW)=Body weight (day X)/Body weight (the firstday)

On the basis of the volumes of tumors obtained on Day 1, the mice weregrouped such that averages of the tumor volumes were substantially equalamong the groups. The experiment was conducted on groups each consistingof six mice. The test compound was dissolved in DMSO and a solution wasstored in the freezer before use. Immediately before the administration,the stock solution was diluted with saline. The test compound in salinewas intravenously once-weekly administered at 20 μg/kg, 60 μg/kg, or 180μg/kg for 2 weeks (on Day 1 and Day 8). The compound treatment inducedtumor regression at 180 μg/kg and tumor growth delay at 60 μg/kg. Theadministration at 180 μg/kg completely eradicated the xenograft tumorsin all mice on Day 22.

Pharmacological Test Example 18. Effect on CD31-Positive Vessel in theFaDu Subcutaneous Model in Mice (FIG. 13)

A human squamous cell carcinoma of the head and neck (SCCHN) cell lineFaDu, which had been cultured in an RPMI-1640 medium containing 10% FBS,and penicillin-streptomycin (culture medium), was adjusted to aconcentration of 5×10⁷ cells/mL with culture medium to prepare a cellsuspension. The cell suspension was inoculated in a volume of 100 μLinto a subcutaneous part of a right flank of nude mice, 6 weeks of ages(CAnN.Cg-Foxnlnu/CrlCrlj, female, Charles River Laboratories Japan,Inc.). Ten days after cell inoculation, the mice were grouped such thataverages of the tumor volumes were substantially equal among the groups.The experiment was conducted on groups each consisting of 6 mice. Eachtest compound was dissolved in DMSO and a solution was stored in thefreezer before use. Immediately before the administration, the stocksolution was diluted with saline. The test compound in saline wasintravenously administered at 20 μg/kg, 60 μg/kg, or 180 μg/kg. Fivedays after the single administration, tumor samples were collected andfixed with IHC Zinc Fixative (BD Pharmingen) at 4° C. for 24 hours.Paraffin-embedded tissues were sectioned (3 μm), mounted on positivelycharged slides, and air-dried. Immunohistochemical staining of CD31 wasconducted using Ventana autostainer model Discover XT (RocheDiagnostics) according to the manufacture's protocol. Sections weredeparaffinized, conditioned and the antigens were retrieved with CC1(Ventana Medical Systems). Slides were blocked with Blocker A andBlocker B (Endogenous biotin blocking kit, Roche Diagnostics). Ratanti-mouse IgG CD31 antibody (Dianova GmbH) was applied at 2 μg/mL.Sections were incubated with the antibody for 6 hours, followed by 32minutes incubation with biotinylated anti-rat IgG antibody (JacksonImmunoResearch Laboratories) at 2.2 μg/mL. The detection was performedwith Streptavidin-HRP D for 16 minutes, followed by incubation with DABD and DAB H₂O₂ D (DABMap kit, Ventana Medical Systems, Inc.) for 8minutes. Slides were counterstained with Hematoxylin II (RocheDiagnostics) for 16 min, followed by incubation with Bluing reagent for4 minutes. Sections were dehydrated in graded ethanols, defatted inxylene replacement and covered with DPX® (Merck KGaA). Immunostainedslides were scanned using Vectra® 2 Automated Slide Imaging System(Perkin Elmer Inc.). The number of blood vessels in the whole tumor wasquantified by counting the CD31-positive objects using inform 2 software(PerkinElmer Inc.) Area of the tumor region was measured by assessingthe hematoxylin-staining area using inform 2 software (PerkinElmer Inc.)The number of blood vessels was normalized by the area of the tumorregion. The single administration of test compound at doses of 20, 60,and 180 μg/kg increased the tumor blood vessel number. The ratios ofblood vessel number in the test compound-dosing groups compared tonon-treat group were calculated with the below formula:

Tumor vessel ratio=blood vessel number of the test compound-dosinggroup/blood vessel number of the non-treat group)

Pharmacological Test Example 19. Effect on α-SMA-Positive CAFs in theFaDu Subcutaneous Model in Mice (FIG. 14)

A human squamous cell carcinoma of the head and neck (SCCHN) cell lineFaDu, which had been cultured in an RPMI-1640 medium containing 10% FBS,and penicillin-streptomycin (culture medium), was adjusted to aconcentration of 5×10⁷ cells/mL with culture medium to prepare a cellsuspension. The cell suspension was inoculated in a volume of 100 μLinto a subcutaneous part of a right flank of nude mice, 6 weeks of ages(CAnN.Cg-Foxnlnu/CrlCrlj, female, Charles River Laboratories Japan,Inc.). Ten days after cell inoculation, the mice were grouped such thataverages of the tumor volumes were substantially equal among the groups.The experiment was conducted on groups each consisting of 5 mice. Eachtest compound was dissolved in DMSO and a solution was stored in thefreezer before use. Immediately before the administration, the stocksolution was diluted with saline. The test compound in saline wasintravenously administered at 20 μg/kg, 60 μg/kg, or 180 μg/kg. Two daysor 5 days after the single administration, tumor samples were collectedand fixed with IHC Zinc Fixative (BD Pharmingen) at 4° C. for 24 hours.Paraffin-embedded tissues were sectioned (3 μm), mounted on positivelycharged slides, and air-dried. Sections were deparaffinized, conditionedand the antigens were retrieved using microwave with 1 mM EDTA at pH6.0. Sections were blocked with 1% of BSA in TBS. Mouse anti-α-SMAmonoclonal antibody conjugated with alkaline phosphatase (clone 1A4,Sigma) was applied at 5 μg/mL. Sections were incubated with the antibodyfor 2.5 hr. The detection was performed with Fast red II substrate kit(Nichirei Bioscience Inc.). Sections were counterstained with Mayer'sHematoxylin (Muto Pure Chemicals) for 50 seconds. Sections weredehydrated in graded ethanols, defatted in xylene replacement andcovered with DPX (Merck KGaA). Immunostained slides were scanned usingVectra 2 Automated Slide Imaging System (Perkin Elmer Inc.). The area ofα-SMA-positive region in the whole tumor was quantified by counting theα-SMA-positive objects using inform 2 software (PerkinElmer Inc.) Areaof the tumor region was measured by assessing the hematoxylin-stainingarea using inform 2 software (PerkinElmer Inc.). The area of theα-SMA-positive region was normalized by the area of the tumor region.The single administration of test compound significantly reduced theα-SMA positive area at doses of 60 and 180 μg/kg on Day 3 and at a doseof 180 μg/kg on Day 6. A suppression rate of the α-SMA-positive area ofthe test compound-dosing group was calculated with the below formula:

α-SMA ratio=α-SMA area of the test compound-dosing group/α-SMA area ofthe non-treat group

Pharmacological Test Example 20. Effects on Tenascin-C andEDA-Fibronectin in the FaDu Subcutaneous Model in Mice (FIG. 15)

A human squamous cell carcinoma of the head and neck (SCCHN) cell lineFaDu, which had been cultured in an RPMI-1640 medium containing 10% FBS,and penicillin-streptomycin (culture medium), was adjusted to aconcentration of 5×10⁷ cells/mL with culture medium to prepare a cellsuspension. The cell suspension was inoculated in a volume of 100 μLinto a subcutaneous part of a right flank of nude mice, 6 weeks of ages(CAnN.Cg-Foxnlnu/CrlCrlj, female, Charles River Laboratories Japan,Inc.). Ten days after cell inoculation, the mice were grouped such thataverages of the tumor volumes were substantially equal among the groups.The experiment was conducted on groups each consisting of 5 mice.Compound (1) was dissolved in DMSO and a solution was stored in thefreezer before use. Compound (1) (180 μg/kg) and Cetuximab (CTX,Erbitux®, Merck Serono Co. Ltd.) (10 mg/kg) was diluted with saline andintravenously injected on Day 1. Five days after the singleadministration, tumor samples were collected and fixed with IHC ZincFixative (BD Pharmingen) at 4° C. for 24 hr. Paraffin-embedded tissueswere sectioned (3 μm), mounted on positively charged slides, andair-dried. Sections were deparaffinized, conditioned and the antigenswere retrieved using microwave with 1 mM EDTA at pH 6.0 for Tenascin-C.For EDA-fibronectin, the antigens retrieval procedure was not necessary.Sections were incubated with BLOXALL Blocking Solution (Vector Labs) for10 min to block endogenous peroxidase, and with Mouse on Mouse IgBlocking Reagent (Vector Labs) for 1 hour, and then with 2.5% normalhorse serum for 30 minutes. For immunohistochemical staining ofTenascin-C, mouse anti-Tenascin-C monoclonal antibody (clone 4C8MS, IBL)was applied at 5 μg/mL. Sections were incubated with the antibodyovernight at 4° C. For immunohistochemical staining of EDA-fibronectin,mouse anti-EDA-fibronectin monoclonal antibody (clone IST-9, Abcam) wasapplied at 1.5 μg/mL. Sections were incubated with the antibody for 1hour at room temperature. The detection was performed with Mouse OnMouse ImmPRESS™ Peroxidase Polymer Kit (Vector Labs). Sections werecounterstained with Mayer's Hematoxylin (Muto Pure Chemicals) for 50sec. Sections were dehydrated in graded ethanols, defatted in xylenereplacement and covered with DPX (Merck KGaA). Immunostained slides werescanned using Vectra 2 Automated Slide Imaging System (Perkin ElmerInc.). The expression levels of both Tenascin-C and ED-A fibronectinwere reduced in the Compound (1) and CTX treated tumors compared withcontrol tumors.

Pharmacological Test Example 21. Antitumor Effects in FaDu SubcutaneousXenograft Model in Combination with Cetuximab in Mice (FIG. 16)

A human squamous cell carcinoma of the head and neck (SCCHN) cell lineFaDu, which had been cultured in an RPMI-1640 medium containing 10% FBS,and penicillin-streptomycin, was adjusted to a concentration of 5×10⁷cells/mL with Hanks' Balanced Salt Solution to prepare a cellsuspension. The cell suspension was inoculated in a volume of 100 L intoa subcutaneous part of a right flank of athymic mice(CAnN.Cg-Foxnlnu/CrlCrlj, female, 7 weeks old, Charles River JapanInc.). Ten days after cell inoculation (Day 1), the length and the widthof a tumor in each mouse were measured by using an electronic digitalcaliper (Mitutoyo Corporation), so as to calculate the volume of thetumor in accordance with the following calculation formula:

Tumor volume (mm³)=Longest diameter (mm)×Shortest diameter (mm)×Shortestdiameter (mm)/2

Relative tumor volume (RTV)=Tumor volume (day X)/Tumor volume (the firstday)

On the basis of TV, the mice were randomly grouped (Day 1). Each groupwas consisted in six mice. Compound (1) was dissolved in DMSO and asolution was stored in the freezer before use. Compound (1) (20, 60, or180 μg/kg) and Cetuximab (CTX, Erbitux®, Merck Serono Co., Ltd.) (10mg/kg) was diluted with saline and intravenously injected on Day 1.Changes of RTV of each group were shown in FIG. 16 . At doses of 180μg/kg and 60 μg/kg, antitumor efficacies of Compound (1) with CTX werestronger than that of CTX monotherapy with tumor regression. Theantitumor efficacy of Compound (1) at doses of 20 μg/kg in combinationwith CTX tended to be stronger than that of CTX monotherapy.

Pharmacological Test Example 22. Antitumor Effects in the Soft TissueSarcoma Xenograft Models in Mice as Monotherapy (FIG. 17) MES-SA

A human uterine sarcoma cell line MES-SA, which had been cultured in anRPMI-1640 containing 10% FBS, and penicillin-streptomycin, was adjustedto a concentration of 2×10⁸ cells/mL with Hanks' Balanced Salt Solutionto prepare a cell suspension, and the suspension was mixed with Geltrex®(Thermo Fisher Scientific Inc., #A1413202) in a ratio of 1:1 to preparea cell suspension in a concentration of 1×10¹ cells/mL. The cellsuspension was inoculated in a volume of 100 μL into a subcutaneous partof a right flank of nude mice, 6 weeks of ages (CAnN.Cg-Foxnlnu/CrlCrlj,female, Charles River Laboratories Japan Inc.). Six days after cellinoculation (Day 1), the shortest diameter and the longest diameter of atumor in each mouse were measured by using an electronic digital caliper(Digimatic™ caliper, Mitutoyo Corporation), so as to calculate thevolume of the tumor in accordance with the following calculationformulae:

Tumor volume (mm³)=Longest diameter (mm)×Shortest diameter (mm)×Shortestdiameter (mm)/2

Relative tumor volume (RTV)=Tumor volume (day X)/Tumor volume (the firstday)

On the basis of the volumes of tumors obtained on Day 1, the mice weregrouped such that averages of the tumor volumes were substantially equalamong the groups. The experiment was conducted on groups each consistingof 6 mice. The test compound was dissolved in DMSO and a solution wasstored in the freezer before use. Immediately before the administration,the stock solution was diluted with saline. The test compound in salinewas intravenously once-weekly administered at 180 μg/kg for 2 weeks (onDay 1 and Day 8). The antitumor activity was observed with tumor growthdelay in the treated group.

HT-1080

A human fibrosarcoma cell line HT-1080, which had been cultured in anE-MEM containing 10% FBS, NEAA and antibiotics was adjusted to aconcentration of 3×10⁷ cells/mL with medium to prepare a cellsuspension. The cell suspension was inoculated in a volume of 100 μLinto a subcutaneous part of a right flank of athymic mice(CAnN.Cg-Foxnlnu/CrlCrlj, female, 6 weeks old, Charles River JapanInc.). Six days after cell inoculation (Day 1), the length and the widthof a tumor in each mouse were measured by using an electronic digitalcaliper (Mitutoyo Corporation), so as to calculate the volume of thetumor in accordance with the following calculation formula:

Tumor volume (mm³)=Longest diameter (mm)×Shortest diameter (mm)×Shortestdiameter (mm)/2

Relative tumor volume (RTV)=Tumor volume (day X)/Tumor volume (the firstday)

On the basis of TV, the mice were randomly grouped (Day 1). Each groupwas consisted in six mice. Compound (1) was dissolved in DMSO and asolution was stored in the freezer before use. Compound (1) (180 μg/kg)was diluted with saline and intravenously injected on Day 1 and Day 8.Changes of RTV of each group was shown in FIG. 17 . The antitumoractivity was observed with tumor regression in the treated group.

CTG-2041

Tumor fragments of human angiosarcoma CTG-2041 were implanted s.c. inthe left flank of female mice. Tumor growth was monitored twice a weekusing digital caliper, so as to calculate the volume of the tumor inaccordance with the following calculation formula:

Tumor volume (mm³)=Longest diameter (mm)×Shortest diameter (mm)×Shortestdiameter (mm)/2

Relative tumor volume (RTV)=Tumor volume (day X)/Tumor volume (the firstday)

When the volume of tumors reached approximately 200 mm³, animals arematched by tumor volume into treatment or control groups and dosinginitiated on Day 1. Each group was consisted with five mice. Compound(1) was dissolved in DMSO and a solution was stored in the freezerbefore use. Compound (1) (100 μg/kg) diluted in saline and intravenouslyinjected on Day 1 and Day 8. Changes of RTV of each group were shown inFIG. 17 . The antitumor activity was observed with tumor regression inthe treated group.

Pharmacological Test Example 23. Antitumor Effects in the EndometrialCancer Sarcoma Xenograft Models in Mice as Monotherapy (FIG. 18) HEC-108

A human endometrial cancer cell line HEC-108, which had been cultured inan E-MEM containing 15% FBS and antibiotics were adjusted to aconcentration of 7.14×10⁷ cells/mL with medium to prepare a cellsuspension. The cell suspension was inoculated in a volume of 150 μLinto a subcutaneous part of a right flank of athymic mice(CAnN.Cg-Foxnlnu/CrlCrlj, female, 6 weeks old, Charles River JapanInc.). Thirteen days after cell inoculation (Day 1), the length and thewidth of a tumor in each mouse were measured by using an electronicdigital caliper (Mitutoyo Corporation), so as to calculate the volume ofthe tumor in accordance with the following calculation formula:

Tumor volume (mm³)=Longest diameter (mm)×Shortest diameter (mm)×Shortestdiameter (mm)/2

Relative tumor volume (RTV)=Tumor volume (day X)/Tumor volume (the firstday)

On the basis of TV, the mice were randomly grouped (Day 1). Each groupwas consisted in six mice. Compound (1) was dissolved in DMSO and asolution was stored in the freezer before use. Compound (1) (180 μg/kg)was diluted in saline and intravenously injected on Day 1 and Day 8.Changes of RTV of each group was shown in FIG. 18 . The antitumoractivity was observed with tumor growth delay in the treated group.

AN3CA

A human endometrial cancer cell line AN3CA, which had been cultured inan E-MEM containing 10% FBS, and penicillin-streptomycin, was adjustedto a concentration of 1.4×10⁸ cells/mL with Hanks' Balanced SaltSolution to prepare a cell suspension, and the suspension was mixed withGeltrex® (Thermo Fisher Scientific Inc., #A1413202) in a ratio of 1:1 toprepare a cell suspension in a concentration of 7×10⁷ cells/mL. The cellsuspension was inoculated in a volume of 100 μL into a subcutaneous partof a right flank of nude mice, 6 weeks of ages (CAnN.Cg-Foxnlnu/CrlCrlj,female, Charles River Laboratories Japan Inc.). Twelve days after cellinoculation (Day 1), the shortest diameter and the longest diameter of atumor in each mouse were measured by using an electronic digital caliper(Digimatic™ caliper, Mitutoyo Corporation), so as to calculate thevolume of the tumor in accordance with the following calculationformulae:

Tumor volume (mm³)=Longest diameter (mm)×Shortest diameter (mm)×Shortestdiameter (mm)/2

Relative tumor volume (RTV)=Tumor volume (day X)/Tumor volume (the firstday)

On the basis of the volumes of tumors obtained on Day 1, the mice weregrouped such that averages of the tumor volumes were substantially equalamong the groups. The experiment was conducted on groups each consistingof five mice. The test compound was dissolved in DMSO and a solution wasstored in the freezer before use. Immediately before the administration,the stock solution was diluted with saline. The test compound in salinewas intravenously once-weekly administered at 180 μg/kg for 2 weeks (onDay 1 and Day 8). The antitumor activity was observed with tumorregression in the treated group.

Pharmacological Test Example 24. Growth Inhibition in NCI-H23, HCC1954,and MES-SA Cell Lines NCI-H23 Growth Inhibition Assay

In this assay, the growth inhibitory activities of Compound D-6 (Example26), E-2, G-4, H-2, 1-2, J-1, K-2, L-6, M-3, and N-1 in a human lungcancer cell line NCI-H23 were measured. NCI-H23 cells were maintained inRPM-1640 (Wako Pure Chemical Industries, Ltd., 187-02021) mediumcontaining 10% fetal bovine serum (FBS: Sigma, 172012), and penicillinand streptomycin (Wako Pure Chemical Industries, Ltd., 168-23191) in a5% CO₂ incubator (37° C.). To each well of a 96 well plate (Becton,Dickinson and Company, 353219), 75 μL of NCI-H23 cell suspensionadjusted to a concentration of 4×10⁴ cells/mL with the culture mediumwas added, and the cells were incubated overnight in a 5% CO₂ incubator(37° C.). On the next day, 25 μL of each compound in three-fold dilutionseries suspended in the culture medium was added to each well, and theresultant was incubated for 3 days in a 5% CO₂ incubator (37° C.). Then,cell viability was determined by CellTiter-Glo® Luminescent CellViability Assay (Promega) with EnVision 2103 Multilabel Reader(Perkin-Elmer, Wellesley, Mass.). Value of the wells containing cellswithout adding the test substance was defined as 100% and the value ofthe wells containing no cells was defined as 0%. The concentration ofthe test substance necessary for inhibiting the cell growth by 50%(i.e., an IC₅₀ value) was calculated, and summarized in Table 14.

HCC1954 Growth Inhibition Assay

In this assay, the growth inhibitory activities of Compound D-6 (Example26), E-2, G-4, H-2, I-2, J-1, K-2, L-6, M-3, and N-1 in a human breastcancer cell line HCC1954 were measured. HCC1954 cells were maintained inRPMI-1640 medium modified to contain 2 mM L-glutamine, 10 mM HEPES, 1 mMsodium pyruvate, 4500 mg/L glucose, and 1500 mg/L sodium bicarbonate(ATCC 30-2001) containing 10% fetal bovine serum (FBS: Sigma, 172012),and penicillin and streptomycin (Wako Pure Chemical Industries, Ltd.,168-23191) in a 5% CO₂ incubator (37° C.). To each well of a 96 wellplate (Becton, Dickinson and Company, 353219), 75 μL of HCC1954 cellsuspension adjusted to a concentration of 4×10⁴ cells/mL with theculture medium was added, and the cells were incubated overnight in a 5%CO₂ incubator (37° C.). On the next day, 25 μL of each compound inthree-fold dilution series suspended in the culture medium was added toeach well, and the resultant was incubated for 3 days in a 5% CO₂incubator (37° C.). Then, cell viability was determined byCellTiter-Glo® Luminescent Cell Viability Assay (Promega) with EnVision2103 Multilabel Reader (Perkin-Elmer, Wellesley, Mass.). Value of thewells containing cells without adding the test substance was defined as100% and the value of the wells containing no cells was defined as 0%.The concentration of the test substance necessary for inhibiting thecell growth by 50% (i.e., an IC₅₀ value) was calculated, and summarizedin Table 14.

MES-SA Growth Inhibition Assay

In this assay, the growth inhibitory activities of Compound D-6 (Example26), E-2, G-4, H-2, 1-2, J-1, K-2, L-6, M-3, and N-1 in a human lungcancer cell line MES-SA were measured. MES-SA cells were maintained inRPM-1640 (Wako Pure Chemical Industries, Ltd., 187-02021) mediumcontaining 10% fetal bovine serum (FBS: Sigma, 172012), and penicillinand streptomycin (Wako Pure Chemical Industries, Ltd., 168-23191) in a5% CO₂ incubator (37° C.). To each well of a 96 well plate (Becton,Dickinson and Company, 353219), 75 μL of MES-SA cell suspension adjustedto a concentration of 4×10⁴ cells/mL with the culture medium was added,and the cells were incubated overnight in a 5% CO₂ incubator (37° C.).On the next day, 25 μL of each compound in three-fold dilution seriessuspended in the culture medium was added to each well, and theresultant was incubated for 3 days in a 5% CO₂ incubator (37° C.). Then,cell viability was determined by CellTiter-Glo® Luminescent CellViability Assay (Promega) with EnVision 2103 Multilabel Reader(Perkin-Elmer, Wellesley, Mass.). Value of the wells containing cellswithout adding the test substance was defined as 100% and the value ofthe wells containing no cells was defined as 0%. The concentration ofthe test substance necessary for inhibiting the cell growth by 50%(i.e., an IC₅₀ value) was calculated, and summarized in Table 14.

TABLE 14 Cell growth inhibition IC₅₀ (nM) Compound NCI-H23 HCC1954MES-SA Compound D-6 (Example 26) 0.0220 0.0104 0.0138 Compound E-2 0.5950.0805 0.856 Compound G-4 0.136 0.0335 0.326 Compound H-2 0.286 0.1230.146 Compound I-2 0.103 0.00624 0.0570 Compound J-1 0.00992 0.003080.0102 Compound K-2 0.461 0.0700 0.341 Compound L-6 6.81 0.269 2.79Compound M-3 0.0199 0.0101 0.0141 Compound N-1 1.53 0.0800 2.88

Pharmacological Test Example 25. Molecular Mechanisms of theα-SMA-Positive CAF Reduction Using an In Vitro Culture System

The expression of α-SMA was induced in BJ normal human fibroblasts uponco-cultivation with FaDu cells, and the expression was attenuated bytreatment with A83-01, a potent selective inhibitor of theTGF-β-receptor (FIG. 19A). These results suggested that TGF-β plays amajor role as a mediator of α-SMA induction in this system. In addition,immunofluorescence analysis revealed that treatment with Compound (1)interfered with α-SMA induction by TGF-β in BJ cells without growthinhibitory activity (FIG. 19A, FIG. 20A).

Compound (1) did not significantly change the TGF-β-inducedphosphorylation and nuclear localization of Smad2/3 (FIGS. 20D-20E), butit did reduce the activation of the PI3K/AKT/mTOR pathway, which playsessential roles in TGF-β-induced α-SMA expression (FIGS. 19C and 19F,FIGS. 20B-20C). Moreover, TGF-β treatment enhanced P3-tubulin expressionand microtubule network formation, which were diminished by co-treatmentwith Compound (1) in BJ cells (FIG. 19D). Under the same experimentalconditions, the enhanced formation of focal adhesions, which weredetected as punctate structures with an antibody against phosphorylatedFAK at tyrosine-397, after stimulation with TGF-β was decreased in BJcells by treatment with Compound (1) (FIG. 19E). Many signallingcomplexes are assembled in focal adhesion sites, and those complexesdispatch several downstream signals, including those involved in thePI3K/AKT/mTOR pathway. See, e.g., Sulzmaier et. al. “FAK in cancer:mechanistic findings and clinical applications”, Nature Reviews Cancer2014, 14, 598-610. In fact, treatment with defactinib, an FAK inhibitor,clearly decreased the level of α-SMA expression as well as S6 ribosomalprotein phosphorylation induced by TGF-β in BJ cells (FIGS. 19G-19H).All of the abovementioned phenomena were also observed in other normalhuman fibroblasts (TIG3 cells) (FIGS. 21A-21H).

Pharmacological Test Example 26. Bladder Carcinoma Cell Lines GrowthInhibition Assay

In this assay, the growth inhibitory activity of Compound (1) in humanbladder carcinoma cell lines RT4, RT112/84, and UM-UC-3 was measured.RT4 and RT112/84 cells were maintained in a RPMI-1640 (FUJIFILM WakoPure Chemical Corporation, 189-02025) medium containing 10% fetal bovineserum (FBS: SIGMA Life Science, 172012-500ML), and penicillin andstreptomycin (FUJIFILM Wako Pure Chemical Corporation, 168-23191) in a5% CO₂ incubator (37° C.). UM-UC-3 cells were maintained in an E-MEM(FUJIFILM Wako Pure Chemical Corporation, 051-07615) medium containing10% FBS, 1 mM Sodium Pyruvate (FUJIFILM Wako Pure Chemical Corporation,190-14881), and penicillin and streptomycin in a 5% CO₂ incubator (37°C.). To each well of a 96 well plate (Corning, 3904), 90 μL of each cellsuspension adjusted to a concentration of 5.55×10³ cells/mL (RT4 orUM-UC-3) or 3.33×10⁴ cells/mL (RT112/84) with the culture medium wasadded, and the cells were incubated overnight in a 5% CO₂ incubator (37°C.). On the next day, 10 μL of Compound (1) in three-fold dilutionseries suspended in the culture medium was added to each well, and theresultant was incubated for 3 days in a 5% CO₂ incubator (37° C.). Then,cell viability was determined by CellTiter-Glo® 2.0 Assay (PromegaCorporation, G9243) with ARVO-X4 Multimode Plate Reader (Perkin-Elmer,Wellesley, Mass.). Value of the wells containing cells without addingthe test compounds was defined as 100% and the value of the wellscontaining no cells was defined as 0%. The concentrations of the testcompound necessary for inhibiting the cell growth by 50% (i.e., an IC₅₀value) was calculated, and shown in Table 15.

TABLE 15 (IC₅₀ (nM)) Test compound RT4 RT112/84 UM-UC-3 Compound (1)0.110 0.0497 0.512

Pharmacological Test Example 27. Antitumor Effect in the HS-SY-IIXenograft Model in Mice as Monotherapy (FIG. 22)

A human synovial sarcoma cell line HS-SY-II, which had been cultured ina DMEM containing 10% FBS, and penicillin-streptomycin, was adjusted toa concentration of 1×10¹ cells/mL with 50% Geltrex (Thermo FisherScientific) in Hanks' Balanced Salt Solution to prepare a cellsuspension. The cell suspension was inoculated in a volume of 100 μLinto a subcutaneous part of a right flank of nude mice, 5 weeks of ages(CAnN.Cg-Foxnlnu/CrlCrlj, female, Charles River Laboratories JapanInc.). Thirty days after cell inoculation (Day 1), the shortest diameterand the longest diameter of a tumor in each mouse were measured by usingan electronic digital caliper (Digimatic™ caliper, MitutoyoCorporation), so as to calculate the volume of the tumor in accordancewith the following calculation formulae:

Tumor volume (mm³)=Longest diameter (mm)×Shortest diameter (mm)×Shortestdiameter (mm)/2

Relative tumor volume (RTV)=Tumor volume (day X)/Tumor volume (the firstday)

Relative body weight (RBW)=Body weight (day X)/Body weight (the firstday)

On the basis of the volumes of tumors obtained on Day 1, the mice weregrouped such that averages of the tumor volumes were substantially equalamong the groups. The experiment was conducted on groups each consistingof six mice. Compound (1) was dissolved in DMSO and a solution wasstored in the freezer before use. Immediately before the administration,the stock solution was diluted with saline. The test compound in salinewas intravenously once-weekly administered at 90 μg/kg or 180 μg/kg for2 weeks (on Day 1 and Day 8). The tumor growth delay was observed in 90μg/kg-treated group and the tumor regression was observed in 180μg/kg-treated group.

Pharmacological Test Example 28. Antitumor Effect in the HuTu 80Xenograft Model in Mice as Monotherapy (FIG. 23)

A human duodenal cell line HuTu 80, which had been cultured in a EMEMcontaining 10% FBS, and penicillin-streptomycin, was adjusted to aconcentration of 3×10⁷ cells/mL in PBS to prepare a cell suspension. Thecell suspension was inoculated in a volume of 100 μL into a subcutaneouspart of a right flank of nude mice, 7 weeks of ages(CAnN.Cg-Foxnlnu/CrlCrlj, female, Charles River Laboratories JapanInc.). Seven days after cell inoculation (Day 1), the shortest diameterand the longest diameter of a tumor in each mouse were measured by usingan electronic digital caliper (Digimatic™ caliper, MitutoyoCorporation), so as to calculate the volume of the tumor in accordancewith the following calculation formulae:

Tumor volume (mm³)=Longest diameter (mm)×Shortest diameter (mm)×Shortestdiameter (mm)/2

Relative tumor volume (RTV)=Tumor volume (day X)/Tumor volume (the firstday)

Relative body weight (RBW)=Body weight (day X)/Body weight (the firstday)

On the basis of the volumes of tumors obtained on Day 1, the mice weregrouped such that averages of the tumor volumes were substantially equalamong the groups. The experiment was conducted on groups each consistingof six mice. Compound (1) was dissolved in DMSO and a solution wasstored in the freezer before use. Immediately before the administration,the stock solution was diluted with saline. The test compound in salinewas intravenously once-weekly administered at 45, 90, or 180 μg/kg for 2weeks (on Day 1 and Day 8). The tumor growth delay was observed in 45and 90 μg/kg-treated group and the tumor regression was observed in 180μg/kg-treated group. Tumor regression (%) for Compound (1) is shown inTable 16.

TABLE 16 Dose Tumor Regression Test compound (mg/kg) (%) Compound (1)0.18 99

EQUIVALENTS AND SCOPE

In the claims articles such as “a,” “an,” and “the” may mean one or morethan one unless indicated to the contrary or otherwise evident from thecontext. Claims or descriptions that include “or” between one or moremembers of a group are considered satisfied if one, more than one, orall of the group members are present in, employed in, or otherwiserelevant to a given product or process unless indicated to the contraryor otherwise evident from the context. The invention includesembodiments in which exactly one member of the group is present in,employed in, or otherwise relevant to a given product or process. Theinvention includes embodiments in which more than one, or all of thegroup members are present in, employed in, or otherwise relevant to agiven product or process.

Furthermore, the invention encompasses all variations, combinations, andpermutations in which one or more limitations, elements, clauses, anddescriptive terms from one or more of the listed claims is introducedinto another claim. For example, any claim that is dependent on anotherclaim can be modified to include one or more limitations found in anyother claim that is dependent on the same base claim. Where elements arepresented as lists, e.g., in Markush group format, each subgroup of theelements is also disclosed, and any element(s) can be removed from thegroup. It should it be understood that, in general, where the invention,or aspects of the invention, is/are referred to as comprising particularelements and/or features, certain embodiments of the invention oraspects of the invention consist, or consist essentially of, suchelements and/or features. For purposes of simplicity, those embodimentshave not been specifically set forth in haec verba herein. It is alsonoted that the terms “comprising” and “containing” are intended to beopen and permits the inclusion of additional elements or steps. Whereranges are given, endpoints are included. Furthermore, unless otherwiseindicated or otherwise evident from the context and understanding of oneof ordinary skill in the art, values that are expressed as ranges canassume any specific value or sub-range within the stated ranges indifferent embodiments of the invention, to the tenth of the unit of thelower limit of the range, unless the context clearly dictates otherwise.

Those skilled in the art will recognize or be able to ascertain using nomore than routine experimentation many equivalents to the specificembodiments described herein. The scope of the present embodimentsdescribed herein is not intended to be limited to the above Description,but rather is as set forth in the appended claims. Those of ordinaryskill in the art will appreciate that various changes and modificationsto this description may be made without departing from the spirit orscope of the present invention, as defined in the following claims.

1-5. (canceled)
 6. A compound of Formula (III):

or a pharmaceutically acceptable salt or stereoisomer thereof, wherein:R^(O) is hydrogen, optionally substituted alkyl, optionally substitutedacyl, or an oxygen protecting group; R^(P1) is hydrogen, optionallysubstituted alkyl, optionally substituted acyl, or an oxygen protectinggroup; R^(X) is hydrogen or —OR^(Xa); R^(Y) is hydrogen or —OR^(Ya);R^(Xa) and R^(Ya) are independently hydrogen, optionally substitutedalkyl, optionally substituted acyl, or an oxygen protecting group,optionally wherein R^(Xa) and R^(Ya) are joined together with theintervening atoms to form optionally substituted heterocyclyl; and n is1 or
 2. 7. The compound of claim 6, wherein the compound is of thefollowing formula:

or a pharmaceutically acceptable salt or stereoisomer thereof.
 8. Thecompound of claim 6 or 7, provided that the compound is not thefollowing:

or a pharmaceutically acceptable salt thereof. 9-10. (canceled)
 11. Thecompound of claim 6, or a pharmaceutically acceptable salt orstereoisomer thereof, wherein n is
 1. 12. The compound of claim 6, or apharmaceutically acceptable salt or stereoisomer thereof, wherein n is2. 13-16. (canceled)
 17. The compound of claim 6, or a pharmaceuticallyacceptable salt or stereoisomer thereof, wherein R^(O) is hydrogen. 18.The compound of claim 6, or a pharmaceutically acceptable salt orstereoisomer thereof, wherein R^(O) is optionally substituted C₁₋₆alkyl. 19-22. (canceled)
 23. The compound of claim 6, or apharmaceutically acceptable salt or stereoisomer thereof, wherein R^(P1)is hydrogen. 24-29. (canceled)
 30. The compound of claim 6, or apharmaceutically acceptable salt or stereoisomer thereof, wherein R^(X)and R^(Y) are hydrogen.
 31. The compound of claim 6, or apharmaceutically acceptable salt or stereoisomer thereof, wherein R^(X)and R^(Y) are —OH.
 32. The compound of claim 6, or a pharmaceuticallyacceptable salt or stereoisomer thereof, wherein R^(X) is hydrogen; andR^(Y) is —OH. 33-36. (canceled)
 37. The compound of claim 6, wherein thecompound is selected from the group consisting of:

and pharmaceutically acceptable salts thereof.
 38. The compound of claim6, wherein the compound is selected from the group consisting of:

and pharmaceutically acceptable salts thereof.
 39. (canceled)
 40. Apharmaceutical composition comprising a compound of claim 6, or apharmaceutically acceptable salt or stereoisomer thereof, and apharmaceutically acceptable carrier. 41-44. (canceled)
 45. A method fortreating a proliferative disease in a subject comprising administeringto the subject a compound of claim 6, or a pharmaceutically acceptablesalt or stereoisomer thereof, or a pharmaceutical composition thereof.46. The method of claim 45, wherein the proliferative disease is canceror a tumor.
 47. The method of claim 46, wherein the cancer or tumor ischaracterized by angiogenesis, invasion, or metastasis.
 48. A method forinhibiting growth of tumor or cancer in a subject comprisingadministering to the subject a compound of claim 6, or apharmaceutically acceptable salt or stereoisomer thereof, or apharmaceutical composition thereof. 49-87. (canceled)
 88. The compoundof claim 6, wherein the compound is of the following formula:

or a pharmaceutically acceptable salt or stereoisomer thereof.